ES2559634T3 - Peptides derived from NCAM (FGLs) - Google Patents

Abstract

A peptide of 13 amino acids or less comprising the sequence QQGKSKA, or a pharmaceutically acceptable salt thereof.

Description

Peptides derived from NCAM (FGLs)

Field of the Invention

The present invention relates to new compounds comprising a maximum of 13 contiguous amino acid residues derived from module 11 of fibronectin type 3, of the neural cell adhesion molecule (NCAM), or a variant or fragment thereof, capable of interacting with an FGFR and thus the compounds are capable of inducing differentiation, modulating proliferation, stimulating regeneration, neuronal plasticity and / or cell survival. In addition, the present invention relates to the use of said compounds for the production of a medicament for the treatment of conditions and diseases, where NCAM and / or FGFR play a prominent role.

Background of the invention

NCAM is a cell surface glycoprotein that belongs to the CAM Ig superfamily (for a review see Kiselyov al., 2005). NCAM can be expressed as three main isoforms (A, B, and C) with differences in the cytoplasmic domain. The extracellular part of NCAM is identical for all three isoforms and consists of five modules type 19 and two of type 111 fibronectin (F3). NCAM is widely expressed during embryonic development, while in the adult organism it is found mainly in tissues of neural origin. NCAM plays a major role during the development of the nervous system, mediating adhesion between neural cells and stimulating the spread of neurites and fasciculation. , promoting cell survival and synaptic plasticity (Cremer et al., 1997; Berezin et al., 2000; Bruses and Rutishauser, 2001, Rougon and Hobert, 2003; Walmod et al., 2004). NCAM mediates cell-cell adhesion through homophilic binding and regulates the extension of neurites by FGFR (Doherty and Walsh, 1996; Kiselyov et al., 2003 and 2005). The FGFR site involved in the NCAM binding has been mapped in the Ig3 module, and the corresponding site in NCAM - in the second F3 module (Kiselyov el al., 2003). This interaction leads to the activation of intracellular signaling cascades that mediate cell differentiation and survival.

The identified NCAM fragment that has the sequence EVYVVAENQQGKSKA (FGL peptide) involved in the direct interaction between NCAM and FGFR has recently been suggested as a new candidate compound for the treatment of a variety of pathological disorders where stimulation of FGFR activity can play the key role (WO 03/016351). It is located in F3, 11 and binds to and activates FGF 1 and 2 receptors, promotes neurotgenesis, and in vitro neuronal cell survival (Kiselyev et al., 2003; Neiiendam et al., 2004) and improves long-term memory in rats (Cambon et al., 2004). The FGL peptide has also been shown to promote neuronal survival and rescue cognitive deficit in a rat model of amyloid-beta-induced peptide neurotoxicity (Klementiev et al. 2007)

Summary of the invention

The present invention relates to a compound comprising a maximum of 13 amino acids derived from module 11 of fibronectin type 3, of the neural cell adhesion molecule (NCAM), capable of interacting with an FGFR.

A compound of the invention is capable of inducing differentiation, modulating proliferation, stimulating regeneration, neuronal plasticity and / or cell survival.

Another aspect of the invention relates to the use of a compound of the invention as medicaments and for the preparation of medicaments for the treatment of a condition or disease where FGFR and / or NCAM play a role in the pathology or recovery of a disease.

In addition, the present invention describes a pharmaceutical composition comprising at least one compound of the invention.

Also, in another aspect a compound of the invention can be used for the production of an antibody.

Description of the drawings

Figure 1. Dose-dependent effect of FGLs on FGFR activation. Trex293 cells (Invitrogen) were stably transfected with human FGFR1, a IIlc variant, with a C-terminal Estrepll brand. For the

Determination of phosphorylation, 2> <10 cells were deprived overnight in medium without serum. After treatment with FGLs, at a concentration between 1 and 500 og / ml, or FGF1 (10 ng / ml) for 20 minutes, the cells were lysed and purified phospho-proteins were obtained by using antibodies coupled to antiphosphotyrosine agarose. The purified proteins were separated by SDS-PAGE and transferred to a polyvinylidellO fluoride membrane. Immunoblotting was performed using rabbit antibodies against the recombinant Estrepll brand. Immune complexes were developed and then visualized and quantified using SynGene Gene Tool image analysis software. The results are given as mean values ± DTM from at least three independent experiments and are illustrated as the percentage of control values. • p <0.05 in comparison with the control by t-test for dependent samples.

Figure 2. Effects of FGLs on neurite extension of cerebellar neurons of premature postnatal rat CGCs were prepared from 7-day-old Wistar rats and seeded in an uncoated eight-well Lab-Tek® chamber. The peptide was added to the medium immediately after seeding, and the cells were maintained at 37 ° C, 5% C02 for 24 h. CGC cultures were set at 4% and then immunostained against rat GAP-43. The extent of neurites was determined by image analysis of 200 ± 20 neurons in each individual experiment. The length of neuronal processes per cell was estimated stereologically. The results are given as mean values ± DTM from at least three independent experiments and are illustrated as the percentage of control values. 'p <0.05 compared to the control by t-test for dependent samples.

Figure 3. Effects of a single vehicle administration or FGLs on the short-term social memory evaluated in the social recognition test (SRT). S.c. FGLs (8.0 mglkg) or vehicle (sterile water, 4.0 mllkg) and the rats were tested twice with the initial SRT test performed 1 h and 73 h after administration. The juvenile rat was presented twice with an inter-trial interval of 2 hours. In each experimental group data were obtained from 9 to 11 rats. The results are shown as the mean and DTM of the recognition ratio (RR). A RR value significantly below 0.5 indicates social memory. Comparisons of RR values were made with a hypothetical value of 0.50 using t-test of a sample. ... P <0.01

Figure 4. Effects of a single vehicle administration or FGLs on the short-term social memory evaluated in the social recognition test (SRT). S.c. FGLs (1.0, 2.0, 4.0 and 8.0 mg / kg) or vehicle (sterile water, 4.0 mllkg) and the rats were tested twice with the initial SRT test performed 1 h And 24 h after administration. The juvenile rat was presented twice with an inter-trial interval of 2 hours. In each experimental group data were obtained from 9 to 11 rats. The results are shown as the mean and DTM of the recognition ratio (RR). A RR value significantly below 0.5 indicates social memory. Comparisons of RR values were made with a hypothetical value of 0.50 using t-test of a sample. H P <0.01

Figure S. Effects of subcutaneous administration of FGLs, FGk and VEB1 (2 administrations at the indicated doses) on the short-term social memory evaluated in the social recognition test (SRT). The results are shown as the mean and DTM of the recognition ratio (RR). A RR value significantly below 0.5 indicates social memory Comparisons of RR values with a hypothetical value of 0.50 were performed using t-test of a sample

A: The graph illustrates the results obtained in the SRT 5 hours after the last administration. In each experimental group, results were obtained from 7 to 10 rats. Significant memory enhancing effects were detected in all peptide groups' P <0.05; "P <0.01," · P <0.001.

B: The graph illustrates the results obtained in the SRT 77 hours after the last administration. In each experimental group data were obtained from 8 to 10 rats. Significant memory enhancing effects were detected for peptide groups FGLs, and FGLl "P <0.01

Figure 6. Effects of FGLs on spatial learning in Morris's water maze. The rats received FGLs or vehicle subcutaneously as a single dose of 2.5 mglkg 5, 3 and 1 day before the first trial (trials 1-4 in the graph above). The second trial (trials 5-8 in the previous graph) took place the day after the first trial. The results were analyzed using analysis of variance (ANOVA) and Student's t-test. The data is the average escape time (s) (± DTM) to find the platform during the 2 days of training (t1-t4). 'p <0.05, compared to the vehicle control group at the same time point

Figure 7. Effects of subcutaneous administration of FGLL, FGLs, and VEB1 (2 administrations with an interval of 19 hours, at 8 mgfkg) on scopolamine-induced social amnesia evaluated in the social recognition trial (SRT). The rats were tested with the initial SRT test performed 5 h after the last administration of peptide peptide and 30 minutes after the administration of scopolamine (0.01 mg / kg s.c.). The juvenile rat was presented twice with an inter-trial interval of 15 minutes

A-Recognition ratios (RR) of rats treated with scopolamine and vehicle (Veh) or scopolamine and one of the three peptides as detailed above. The results are shown as the mean and DTM of the recognition ratio (RR). A RR value significantly below 0.5 indicates social memory. Comparisons of RR values were made with a hypothetical value of O, SO using t-test of a sample. In each experimental group data were obtained from 10 rats. Significant effects were detected against scopolamine-induced social amnesia for peptide groups FGLL, and FGLs · P <0.05. B. Cumulative investigation time (T1, in seconds) during the first SRT trial of rats treated with scopolamine and vehicle (Veh) or scopolamine and one of the three peptides as detailed above. The results are shown as the mean and DTM of T1. Comparisons between groups were made by ANOVA followed by Dunnett's multiple comparison test. In each experimental group data were obtained from 10 rats. The

Rats of peptide groups FG ~, and FGLs showed a significantly longer T1 than vehicle-treated rats_P <0.05

Figure 8. The effect of FGLs (administered subcutaneously at the indicated doses), or vehicle on the development of cognitive impairment (short-term social memory deficit) and neurodegeneration after neurointoxication with fragment (25-35) f3-amyloid (A-f3). Comparisons of RR values were made with a hypothetical value of 0.50 using t-test of a sample. Intergroup comparisons were made between Af3 groups using ANOVA of a factor and post-hoc Newman-Keuls test and comparison between vehicle A- [3 and animals operated simulated by a t-test for dependent samples.

A: The effect of the administration of FGLs on the deficit induced by neurotoxicity by f3-amyloid on the performance in the social recognition test The results are shown as the mean and DTM of the recognition ratio (RR). 10-12 animals per groups. A RR value significantly below 0.5 indicates social memory. · P <0.05, or p <0.01 compared to group A- [W.

B: The effect of the administration of FGLs on the decrease in the density of intact neurons induced by [3-amyloid neurotoxicity in the cingulate cortex _ 7-8 rats per group_ + rp <0.05 compared to the group

ApN

C: The effect of the administration of FGLs on the increase in the density of damaged neurons induced by f3-amyloid neurotoxicity in the cingulate cortex. 7-8 rats per group. "p <0.01 +++ r ·· p <0.001 compared to group A- [3N

D: The effect of the administration of FGLs on the increase in amyloid load induced by neurotoxicity by [3

amyloid (% of control value A-I3N) in the cingulate cortex. 7-8 rats per group. Neither the lesion nor the peptide had significant effects

Figure 9_ Effects of intranasal administration of FGLs on neurotoxicity-induced deficiency by [3-amyloid on performance in the social recognition trial. The rats received an inlranasal administration of FGLs at the indicated doses or vehicle daily on days 7 to 21 after Lcy administration of the fragment (25-35) f3-amyloid (A-f3). 8-10 animals per group. The results are expressed as the means and DTM of the recognition ratio and will be analyzed by t-test of a sample: ·· p <0.05.

Figure 10_ Concentration of FGLs in rat blood plasma after intranasal administration of FGLs at 0.2, 0.8 or 3.2 mg / kg daily on days 7 to 21, after i.c.v. of the fragment (25-35) j: Samiloid (A-f3). Blood samples were obtained 30 min and 60 min after the last administration and the concentration of FGLs was measured.

Figure 11. Visual aspect of FGLL and FGLs. 100 mg of FGLL and FGLs, respectively, were diluted in 1 ml of water at room temperature for visual inspection.

Figure 12. Optical microscopy image of FG ~ and FGLs diluted in water and PBS. 5 mg of FG ~ and FGLs were diluted respectively in 1 ml of water or PBS at room temperature A_ 5 mg / ml of FG ~ in water, B_ 5 mg / ml of FGL1 in PBS, C. 5 mg / ml of FGLs in water, D. 5 mg.lml FGLs in PBS.

Figure 13_Light scattering spectra of 90 or 100 mg / ml of FGLL, FGLs in water A_ FGFl, B. FGFs, C water.

Figure 14_ Concentration of FGLs in rat blood plasma after peptide administration_ Rats were admitted to 1.25 mg / kg Lv_ or 2.5 mglkg s_c_ Blood samples were taken at different time points after administration and measured the concentration of FGLs.

Detailed description of the invention

The present invention describes truncated forms of FG ~ with an improved effect, which is more soluble and thus easier to formulate. The new compounds according to the invention comprise a maximum of 13 amino acids derived from the same motif in module 11 of F3 as FGLL.

The elimination of FGLL amino acids from the N-terminal end leads to a more soluble peptide that shows no potential for aggregation, which allows it to be formulated for and administered both intranasally and subcutaneously.

A compound according to the invention comprises a sequence of contiguous amino acids of a maximum of 13 amino acids that is obtained from module II of fibronectin type 3, of NCAM, or a variant or fragment thereof.

In a preferred embodiment, the compounds according to the invention may comprise a contiguous amino acid sequence comprising a maximum of 13 amino acids that is obtained from NCAM_Therefore,

In this embodiment, the amino acid sequence according to the invention can be selected from the following amino acid sequences.

VAENQQGKSKA SEC ID No. 1

EWAENQOGKSKA SEC ID No. 2

NWAENQOGKSKA SEC ID No. 3

NSVAENOOGKSKA SEC ID No. 4

In the present context the conventional one-letter code for amino acid residues is applied as well as the conventional three-letter code. Abbreviations for amino acids are in accordance with the recommendations in the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J. Biochem, 1984, vol. 184, p. 9-37 Throughout the description and claims the three letter code or the one letter code for natural amino acids is used. When the L or O form is not specified, it should be understood that the amino acid in question has the natural form L, cf. Pure & Appl.Chem. Vol. (56 (5) p. 595-624 (1984) or the O form, so that the formed peptides may consist of amino acids of L-form, O-form, or a sequence of L-forms and mixed O-forms.

When nothing is specified it should be understood that the C-terminal amino acid of a peptide for use according to the invention exists as the free carboxylic acid, this can also be specified as "-OH". However, the C-terminal amino acid of a peptide for use according to the invention may be an amidated derivative, which is indicated as "-NH2". When nothing else is indicated, the N-terminal amino acid of a polypeptide comprises a free amino group, this can also be specified as "H-"

A peptide, fragment, homologue or variant thereof according to the invention may also comprise one

or several unnatural amino acids.

A preferred peptide according to the invention is an isolated contiguous peptide sequence comprising a maximum of 13 amino acid residues of NCAM type 3 fibronectin module 11. It is understood that all peptides according to the invention comprise at least one amino acid sequence selected from any of

the sequences SEQ ID NO 1-4 or a fragment, variant or homolog thereof.

Thus, some embodiments of the invention may refer to a peptide comprising a fragment of a sequence selected from SEQ ID NOs 1-4. Another embodiment may refer to variants of SEQ ID NOS 1-4. A further embodiment may refer to homologues of SEQ ID NOs 1-4

A variant according to the invention of an amino acid sequence selected from the sequences SEQ ID NO 1-4 may be

i) an amino acid sequence that has at least 75% identity with a selected sequence, such as 76-80% identity, for example 81-85% identity, such as 86-90% identity, for example 91-95% identity, such as 96-99% identity, where identity is defined as a percentage of identical amino acids in said sequence when checked against the selected sequence. The identity between amino acid sequences can be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;

ii) an amino acid sequence having at least 75% positive amino acid matches with a selected sequence, such as 76-80% positive amino acid matches, PO (eg 81-85% positive amino acid matches, such as 86-90% positive amino acid matches, for example 91-95% positive amino acid matches, such as 96-99% positive amino acid matches, where the positive amino acid match is defined as the presence in the same position in two compared sequences of amino acid residues that have similar physical and chemical properties.The preferred positive amino acid matches of the present invention are K to R, E to D, L to M, O to E, I to V, I to L, Aa S, YaW, Ka a, SaT, N to S and O to R;

iii) an amino acid sequence that is identical to a selected sequence, or has at least 75% identity with said sequence, such as 76-80% identity, for example 81-85% identity, such as a 86-90% identity, for example 91-95% identity, such as 96-99% identity, or has at least 75% positive amino acid matches with the selected sequence, such as 76-80 % of positive amino acid matches, for example 81-85% of positive amino acid matches, such as 86-90% of positive amino acid matches, for example 91-95% of positive amino acid matches, such as 96 -99% positive amino acid matches, and comprises other chemical moieties, for example, phosphoryl, sulfur, acetyl, glucosyl moieties

The expression "variant of a peptide sequence" also means that the peptide sequence can be modified, for example by substitution of one or more of the amino acid residues_ Both Lamino acids and D-amino acids can be used. Another modification may comprise derivatives such as esters, sugars, etc., for example methyl and acetyl esters, as well as modifications with polyethylene glycol.

In addition, an amine group of the peptide can be converted to amides, where the acidic part of the amide is a fatty acid.

In another aspect, the variants of the amino acid sequences according to the invention may comprise, within the same variant, or fragments thereof or between different variants, or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently between them. The variants of the complex, or fragments thereof, can therefore comprise conservative substitutions independently of each other, where at least one glycine (Gly) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting in Ala, Val, Leu, and lIe, and independently thereof, variants, or fragments thereof, where at least one alanine (Ala) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Val, Leu, and lIe, and independently thereof, variants, or fragments thereof, where at least one valine (Val) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Leu, and lIe, and independently thereof, variants, or fragments thereof, where the least one leucine (Leu) of said va Riante, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and lIe, and independently thereof, variants, or fragments thereof, where at least one isoleucine ( Ii) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val and Leu, and independently of the same, variants,

or fragments thereof where at least one aspartic acid (Asp) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Glu, Asn, and Gln, and independently thereof, variants, or fragments thereof, where at least one asparagine (Asn) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, Glu, and Gln, and independently of the same, variants, or fragments thereof, where at least one glutamine (Gln) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, Glu, and Asn, and where at least one phenylalanine (Phe) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, and preferably selection between the group of amino acids consisting of Tyr and Trp, and independently thereof, variants, or fragments thereof, where at least one tyrosine (Tyr) of said variants, or fragments thereof is substituted with a selected amino acid. among the group of amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently thereof, variants, or fragments thereof, where at least one arginine (Arg) of said fragment is substituted with an amino acid selected from the group of amino acids consisting of Lys and His, and independently thereof, variants, or fragments thereof, where at least one lysine (Lys) of said variants , or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Arg and His, and independently thereof, variants, or fragments thereof. same, and independently thereof, variants, or fragments thereof, and where at least one proline (Pro) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp, and His, and independently thereof, variants, or fragments thereof, where at least one cysteine (Cys) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids that It consists of Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.

Therefore, it follows that the same variant of a peptide fragment, or fragment of said variant may comprise more than one conservative amino acid substitution between more than one group of conservative amino acids as defined hereinbefore. The expression ~ conservative amino acid substitution ~ is used as a synonym in this document ~ homologous amino acid substitution ~

Conservative amino acid groups are as follows:

A, G (neutral, weakly hydrophobic),

Q, N, S, T (hydrophilic, not charged)

E, D (hydrophilic acids)

10 H, K, R (hydrophilic, basic)

L, P, 1, V, M, F, Y, W (hydrophobic, aromatic) C (cross bond formation)

Conservative substitutions may be introduced at any position of a preferred predetermined peptide for use according to the invention or fragment thereof. However, it may also be desirable to introduce non-conservative substitutions, particularly, a non-conservative substitution in any one or more positions.

A non-conservative substitution leading to the formation of a variant fragment of the peptide for use according to the invention would differ, for example, substantially in polarity, pOI "eg a moiety with a non-polar side chain (Ala, Leu, Pro , Trp, Val, lIe, Leu, Phe or Met) substituted in the place of a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu , Arg, or Lys, or the replacement of a charged or polar moiety in the place of a non-polar one; and / or ii) would differ substantially in its effect on the orientation of the peptide structure such as the substitution of or at the site of Pro or Gly by another residue; and iii) would differ substantially in electric charge, for example the replacement of a negatively charged residue such as Glu or Asp in the place of a positively charged residue such as Lys, His or Arg (and vice versa ); and / or iv) would differ substantially in steric volume, p OI "example the substitution of a bulky residue such as His, Trp, Phe or Tyr in the place of one that has a small side chain, for example Ala, Glyo Ser (and vice versa)

The amino acid substitution can be made, in one embodiment, based on its hydrophobicity and hydrophilicity values and the relative similarity with the amino acid side chain substituents, including the loading, size.

Examples of variants are:

EX1a-X2b-VAENQQGKSKA DX1a-X2a-VAENQQGKSKA

Where X1a is F, A, C, G, Q, L, M, N, S, Y, T, 1, P, V, most preferred X1a is A, G, L, M, V, P, 1.

And X2b is Q, N, S, T, G or is deleted

Specific non-limiting examples of variables are:

EWAENQQGKSKA (SEQ ID NO 2) NWAENQQGKSKA (SEQ ID No. 3) NSVAENQQGKSKA (SEQ ID No. 4) AVAENQQGKSKA (SEQ ID No. 5) GVAENQQGKSKA (SEQ ID NO 6) LVAENQQGKSKA (SEQ ID NO 7) MVAENQQGKSKA (SEQ ID NO 8) W AENQQGKSKA (SEQ ID NO 9) PVAENQQGKSKA (SEQ ID NO 10) IVAENQQGKSKA (SEQ ID No. 11) QAVAENQQGKSKA (SEQ ID NO 12) QGVAENQQGKSKA (SEQ ID NO 13) QLVAENQQGKSKA (SEQ ID NO 14) QMVAENQQGKSKA (SEQ ID NO 15) QWAENQQGKSKA (SEQ ID NO 16) QPVAENQQGKSKA (SEQ ID NO 17) QIVAENQQGKSKA (SEQ ID NO 18) NAVAENQQGKSKA (SEQ ID NO 19) NGVAENQQGKSKA (SEQ ID NO 20) NLVAENQQGKSKA (SEQ ID NO 21) NMVAENQQGKSKA (SEQ ID NO 22) NWAENQQGKSKA (SEQ ID NO 23) NPVAENQQGKSKA (SEQ ID NO 24) NIVAENQQGKSKA (SEQ ID NO 25) SAVAENQQGKSKA (SEQ ID NO 26) SGVAENQQGKSKA (SEQ ID N, '27) SLVAENQQGKSKA (SEQ ID NO 8) SMVAENQQGKSKA (SEQ ID NO 29) SWAENQQGKSKA (SEQ ID NO 30) SPVAENQQGKSKA (SEQ ID NO 31) SIVAENQQGKSKA (SEQ ID NO 32) TAVAENQQGKSKA (SEQ ID NO 33) TGVAENQQGKSKA (SEQ ID NO 34)

TLVAENQQGKSKA (SEQ ID No. 35) TMVAENQQGKSKA (SEQ ID No. 36) lVVAENQQGKSKA (SEQ ID No. 37) TPVAENQQGKSKA (SEQ ID No. 38) TIVAENQQGKSKA (SEQ ID No. 39) GAVAENQQGKSKA (SEQ ID No. 40) GGVAENQQGKSKA (SEQ ID No. 41) GLVAENQQGKSKA (SEQ ID No. 42) GMVAENQQGKSKA (SEQ ID No. 43) GWAENQQGKSKA (SEQ ID No. 44) GPVAENQQGKSKA (SEQ ID No. 45) GIVAENQQGKSKA (SEQ ID No. 46)

In another embodiment, the variant has the following formula

X3a-X3b-X3c-VAENQQGKSKA

Where X3a is F, A, C, G, Q, L, M, N, S, Y, T, 1, P, Vo is deleted, or more preferred X3a is A, G, L, M, V, P, it is deleted, and X3b is O or E, and X3c is A, G, L, M, V, P, I or is deleted.

With the proviso that X3a and X3c cannot both be deleted

Examples of specific variants are:

DAVAENQQGKSKA (SEQ ID NO 47) OGVAENQQGKSKA (SEQ ID NO 48) oLVAENQQGKSKA (SEQ ID NO 49) oMVAENOOGKSKA (SEQ ID NO 50) OWAENQQGKSKA (SEQ ID NO 51) oPVAENQQGKSKA (SEQ ID NO 52) olVAENQQGKSKA (SEQ ID N "53) EAVAENQQGKSKA (SEQ ID NO 54) EGVAENQQGKSKA (SEQ ID NO 55) ELVAENQQGKSKA (SEQ ID NO 56) EMVAENQQGKSKA (SEQ ID NO 57) EWAENQQQKSKA (SEQ ID NO 2) EPVAENQQGKSKA (SEQ ID NO 58) EIVAENQQGKSKA (SEQ ID NO 59) AOVAENQQGKSKA (SEQ ID NO 64) GOVAENQQGKSKA (SEQ ID NO 61) LOVAEMQQGKSKA (SEQ ID NO 62) MDVAENQQGKSKA (SEQ ID NO 63) VDVAENQQGKSKA (SEQ ID NO 64) POVAENQQGKSKA (SEQ ID NO 65) IDVAENQQGKSKA (SEQ ID NO 66) AEVAENQQGKSKA (SEQ ID NO 67) GEVAEMQOQKSKA (SEQ ID NO 68) LEVAENQQGKSKA (SEQ ID NO 69) MEVAENQQGKSKA (SEQ ID NO 70) VEVAENQQGKSKA (SEQ ID NO 71) PEVAENQQGKSKA (SEQ ID NO 72) IEVAENQQGKSKA (SEQ ID NO 73)

In yet another embodiment, the variant has the form

X2a-X2b-VAENQQGKSKA

Where X2a is F, A, C, G, Q, L, M, N, S, Y, T, 1, P, V, or more preferred X2a is A, G, L, M, V, P, 1.

YX2besD, E.

Both fragments and variants of amino acid sequences according to the invention are the functional equivalents of said sequences.

The term "functional equivalent" of an amino acid sequence means, in the present context, a molecule that meets the criteria for a variant or a fragment of said amino acid sequence described above and that is capable of one or more functional activities of said sequence or a compound comprising said sequence. In a preferred embodiment, the functional equivalent of an amino acid sequence according to the invention is capable of binding to and modulating FGFR activity.

The invention relates to both isolated peptides according to the invention and fusion proteins comprising peptides according to the invention.

In one embodiment, the peptide according to the invention is an isolated peptide. By the term "isolated peptide" is meant that the peptide according to the invention is an individual compound and not a part of another compound. The isolated peptide can be used: using any method of recombinant technology or chemical synthesis and separating it from other compounds, or it can be separated from a longer polypeptide or protein by an enzymatic or chemical cleavage method and further separated from other protein fragments. .

An isolated peptide according to the invention may comprise, in another embodiment, an NCAM fragment comprising a contiguous amino acid sequence derived from NCAM, selected from SEQ ID NOS 1-4 or variant thereof In another embodiment, the peptide isolated may consist of one or more of the sequences SEQ ID No 1-4.

Lipophilic group

In one embodiment, the compound according to the invention is provided with at least one lipophilic group, such as a fatty acid.

The lipophilic group can be attached to the peptide as is or to a linker or spacer connected to the peptide or peptides.

In one embodiment of the invention, an amino group of a spacer forms an amide bond with a carboxyl group of the lipophilic substituent.

In one embodiment of the invention, the lipophilic substituent comprises a partial cidopentanophenanthrene skeleton.

or completely hydrogenated.

In one embodiment of the invention, the lipophilic substituent is a straight or branched chain alkyl group. In a embodiment of the invention, the lipophilic substituent is the acyl group of a straight or branched chain fatty acid. In a further preferred embodiment, the lipophilic substituent is an acyl group of formula · C (C = O) - (CH) & .. o-CH3 El '

Specify the junction point.

In a further preferred embodiment, the lipophilic substituent is an acyl group of formula · C (C = O) - (CH) s.26-CH3 El ' Specify the junction point. In a further preferred embodiment, the lipophilic substituent is an acyl group of formula · C (C = O) - (CH) a.20-CH3. He •

specify the junction point

In a further preferred embodiment, the lipophilic binder is an acyl group of formula · C (C = O) - (CH) mCH3. He • specify the junction point, and m is 8, 9. 10,11, 12, 13, 14, 15, 16, 17, 18_ In a preferred embodiment, he adds, the lipophilic substituent comprises linear or branched dicarboxylic acids. In a further preferred embodiment, the lipophilic substituent comprises linear or branched dicarboxylic acids.

of formula · C (C = O) - (CH) e.w C (O) OH. The • specifies the junction point

In a further preferred embodiment, the lipophilic substituent comprises linear or branched dicarboxylic acids. of formula · C (C = O) - (CH) S.2S-C (O) OH. The • specifies the junction point. In a further preferred embodiment, the lipophilic substituent comprises linear or branched dicarboxylic acids.

of formula · C (C = O) - (CH) S.20-C (O) OH. The • specifies the junction point.

In a further preferred embodiment, the lipophilic substituent comprises linear or branched dicarboxylic acids. of formula · C (C = O) - (CH) S.lS-C (O) OH, El • specifies the point of attachment, In a further preferred embodiment, the lipophilic substituent comprises linear or branched dicarboxylic acids.

of formula "C (C = O) - (CHk C (O) OH. The • specifies the point of attachment. n es8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. Production of peptide sequences

The peptide sequences of the present invention can be prepared by any conventional synthetic method, recombinant DNA technologies, enzymatic cleavage of full-length proteins from which the peptide sequences are derived, or a combination of such methods.

Recombinant preparation

Therefore, in one embodiment the peptides of the invention are produced by the use of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-length protein from which the peptide originates can be prepared synthetically by established conventional methods, for example the phosphoamidine method described by Beaucage and Caruthers, 1981, Tetrahedron Lel ! 22: 1859-1869, or the method described by Matlhes et al., 1984, EMBO J. 3: 801-805. According to the phosphoamidine method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, hybridized, ligated and cloned into suitable vectors.

The DNA sequence encoding a peptide can also be prepared by fragmentation of the DNA sequences encoding the corresponding full length protein of peptide origin, using DNase I according to a conventional protocol (Sambrook et al., Molecular cloning: A Laboratory manual. 2 '"ed., CSHL Press, Cold Spring Harbar, NY, 1989). The present invention relates to full-length proteins selected from the protein groups identified above. The DNA encoding the full-length proteins of the invention can be fragmented, as an alternative, using specific restriction endonucleases. DNA fragments are further purified using conventional procedures described in Sambrook et al., Molecular cloning: A Laboratory manual. 2 '"ed., CSHL Press, Cold Spring Harbar , NY, 1989.

The DNA sequence encoding a full-length protein may also be of genomic or cDNA origin, for example obtained by preparing a genomic or cDNA library and selecting DNA sequences encoding all or part of the full length protein by hybridization using synthetic oligonucleotide probes according to conventional techniques (cf. Sambrook et al., Molecular Cloning: A LabOfatory Manual, 28 Ed., Cold Spring Harbor, 1989). The DNA sequence can also be prepared by polymerase chain reaction using specific primers, for example as described in US 4,683,202 or Saiki el al., 1988, Science 239: 487-491.

The DNA sequence is then inserted into a recombinant expression vector, which can be any vector, which can conveniently undergo recombinant DNA procedures. The choice of vector will often depend on the host cell in which it has to be introduced. Therefore, the vector can be an autonomous replication vector, that is, a vector that exists as an exchromromosomal entity, whose replication is independent of chromosomal replication, for example a plasmid. As an alternative, the vector can be one that, when introduced into a host cell, integrates into the genome of the host cell and replicates along with the chromosome or chromosomes into which it has been integrated

In the vector, the DNA sequence encoding a full-length peptide or protein must be functionally connected to a suitable promoter sequence. The promoter can be any DNA sequence, which shows transcriptional activity in the host cell of choice and can be derived from genes encoding homologous or heterologous proteins to the host cell. Examples of promoters suitable for directing transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981, Mol. Cell Biol. 1: 854-864), the MT-1 promoter (metallothionein gene) (Palmiter et al., 1983, Science

222: 809-814) or the main late promoter of adenovirus 2. A promoter suitable for use in insect cells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS LetL 311: 7-11). Promoters suitable for use in yeast host cells include promoters of yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255: 12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1. 419-434) or alcohol dehydrogenase genes (Young et al., 1982, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., Eds., Plenum Press, New York), or TPI1 promoters (document US 4,599,311) or ADH2-4c (Russell et al., 1983, Nature 304: 652--654). Suitable promoters for use in filamentous fungal host cells are, for example, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4: 2093-2099) or the tpiA promoter.

The coding DNA sequence can also be functionally connected to a suitable terminator, such as the human growth hormone terminator (Palmiter al., Op. Cit.) Or (for fungal hosts) the TPI1 promoters (Alber and Kawasaki, op. Cil.) Or ADH3 (McKnight et al., Op. Cil.). The vector may additionally comprise elements such as polyadenylation signals (for example, from SV 40 or the Elb region of adenovirus 5), transcriptional enhancer sequences (e.g., SV 40 enhancer) and translational enhancer sequences (e.g., those encode adenovirus VA RNA)

The recombinant expression vector may additionally comprise a DNA sequence that enables the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the origin of SV 40 replication. The vector may also comprise a selection marker, for example a gene whose product complements a defect in the host cell, such as a gene encoding dihydrofolate reductase (DHFR) or one that confers resistance to a drug, for example neomycin, hydromycin or methotrexate.

The methods used to ligate the AON sequences encoding the full-length peptides or proteins, the promoter and the terminator, respectively, and to insert 10s into suitable vectors containing the information necessary for replication, are well known to those skilled in the art. matter (cf., for example, Sambrook et al., op.cit.).

To obtain recombinant peptides of the invention, the DNA coding sequences can be fused in a useful way with a second peptide coding sequence and a coding sequence of a protease cleavage site, giving a DNA construct encoding the fusion protein, where The sequence encoding the protease cleavage site is positioned between the HBP fragment and the second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells. In one embodiment, said second peptide is selected from the group comprising glutathione-S-reductase, calf thymosin, bacterial thioredoxin or natural or synthetic variants of human ubiquitin, or peptides thereof. In another embodiment, a peptide sequence comprising a protease cleavage site may be the Factor Xa cleavage site, with the amino acid sequence IEGR, enterokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPRlGS, or Acharombacter Iyticus, with the amino acid sequence

XKX

The host cell into which the expression vector is introduced can be any cell that has the ability to express full-length peptides or proteins, and is preferably a eukaryotic cell, such as invertebrate (insect) or vertebrate cells, by example oocytes of Xenopus laevis or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) cell lines. Methods for transfecting mammalian cells and expressing DNA sequences introduced into the cells in, for example, Kaufman and Sharp, J. Mol. Biol. 159, 1982, p. 601-621; Southem and Berg, 1982, J. Mol. Appl. Genet 1: 327- ~ 1, Loyter et al., 1982, Proc. Natl Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14: 725; Corsaro and Pearson, 1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973, Viral. 52: 456; and Neumann et al., 1982, EMBO J. 1: 841-845.

Alternatively, fungal cells (including yeast cells) can be used as host cells. Examples of suitable yeast cells include Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae. Examples of other fungal cells are filamentous fungal cells, for example Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergi llus spp. for protein expression is described in, for example, EP 238 023.

The medium used to grow the cells may be any conventional means suitable for culturing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for culturing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or can be prepared according to published formulas (for example, in catalogs of the American Type Culture Collection)

The full-length peptides or proteins recombinantly produced by the cells can then be recovered from the culture medium by conventional procedures including separation of the host cells from the medium by centrifugation or filtration, precipitation of the protein components of the supernatant.

or filtered by a salt, for example ammonium sulfate, purification by a variety of chromatographic procedures, for example HPLC, Iophilic exchange chromatography, affinity chromatography, or the like.

Synthetic preparation

Methods for synthetic peptide production are well known in the art. Detailed descriptions as well as practical advice for the production of synthetic peptides can be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant GA ed., Oxford University Press, 2002, or in · Pharmaceutical Formulation: Development of Peptides and Proteins , Frokjaer and Hovgaard eds., Taylor and Francis,

1999.

Peptides can be synthesized, for example, using Fmoc chemistry and with Cm-protected cysteines. After purification by reverse phase HPLC, the peptides can be further processed to obtain, for example, cyclic or C-or N-terminal modified isotherms. The methods for blinding and terminal modification are well known in the art and are described in detail in the manuals cited above.

In a preferred embodiment, the peptide sequences of the invention are produced synthetically, in particular, by the sequence-assisted peptide synthesis method (SAPS)

Peptides can be synthesized in batches in a polyethylene container equipped with a polypropylene filter for

5 filtration or in the continuous flow version of the solid phase method of polyamide (Dryland, A. and Sheppard, RC., (1986) J.Chem. Soco Perkin Trans. 1, 125-137.) In a completely peptide synthesizer automated using 9-ftuorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl, (Boe) as a N-{l-amino protecting group and common suitable protecting groups for side chain functionality

10 Multimeric compound

An isolated peptide sequence of the invention can be connected to another peptide sequence isolated by a chemical bond in a fusion protein or the amino acid sequences can be connected to each other through a linker group. In some embodiments, a peptide sequence of the invention can be formulated as a monomer oligomer (multimer), where each monomer is like a peptide sequence defined above. Particularly, multimeric peptides such as dendrimers can form conformational determinants

or groups due to the presence of multiple flexible peptide monomers. In one embodiment, the compound is a dimer, in another embodiment the compound is a trimer or a tetramer. Polymerization such as repetitive sequences or binding to various vehicles are well known in the art, for example lysine structures, such

20 as lysine dendrimers bearing 4 peptides, 8 peptides, 16 peptides, or 32 peptides. Other vehicles may be lipophilic dendrimers, or micelle-like vehicles formed by lipophilic derivatives, or crashed carbon chain polymer conjugates (star type)

Therefore, according to the invention a multimeric compound can be a polymer comprising two or

25 more identical or different peptide sequences of the invention, wherein in a preferred embodiment, at least one of the two or more amino acid sequences is selected from a sequence of a peptide according to this invention.

In some embodiments, the compound may comprise two identical amino acid sequences or the compound may comprise four identical copies of an amino acid sequence.

In other embodiments, the compound may comprise two or more different amino acid sequences, where at least one of the two amino acid sequences is a sequence selected from the peptide sequences shown herein, or fragments or variants thereof. More preferably two or more sequences

35 are selected from the sequences shown in this document.

Examples of compounds of the invention are:

Z-L-Z,

Z-Z-L,

Z-L-L,

L l r I

45 Z-z,

Z-L-Z-L,

'Z

I \

l L

l. I

Z. fifty

A preferred multimeric compound of the invention is a compound where the amino acid sequences are connected to each other through a linker or linker group, such as

) VM! NlQOO ~ OI \ l "'.

H) VA ~ KA-OOti¡.¡.

FGLs

HYVVA9 «lOGKSI <A-OOOH,

· Yvv.w.lClOG1lllAA-COI! M.

:: r_A ~~ (¡K $ KA-cQNI1,

"ySll" ENQOOKSt <A. ~.

VAÉNQ: oK \ iI \ i \ "... r'i) Q1i-l \ -CQNlH •

. ¡¡

V ~ SM

VAENOQGK \ iiW'10) /! Lo1a-MXlNHo

V: ~~ '"

Where Z1a is one of two amino acids selected from the group F, A, G, a, L, M, N, S, Y, T, 1, P, V Most preferred:

Z1a is one or two amino acids selected from the group A, G, Q, L, N, S, T, 1, P, V _

Even more preferred ·

Z1a is one or two amino acids selected from A, G, S, N Z1a is an amino acid selected from the group F, A, G, a, L, M, N, S, Y, T, 1, P, V More Preferred · 30 Z1a is an amino acid selected from the group A, G, Q, L, N, S, T, 1, P, V. Even more preferred: Z1a is an amino acid selected from A, G, S, N

In one embodiment, two individual peptide sequences of a compound are linked by a linker. A

linker molecule can be selected, according to the invention, from dio, tri- or tetracarboxylic acids

aquirales, said acids having the general formula

or "C · I X ....... (AI,

I

(Blm 'C ·

5 g

XIIA) nCOOHIIIB) mCOOH]

The compound can be produced by the LPA method as described in WO 00f18791.

In one embodiment

n and m are independently an integer from 1 to 20,

15 X is HN, H2N (CR2) pCR, RHN (CR2) pCR, HO (CR2) pCR, HS (CR2) pCR, halogen- (CR2) pCR, HOOC (CR2) pCR, ROOC (CRI) pCR, HCO ( CR2) pCR, RCO (CR2) pCR, [HOOC (A) n) [HOOC (B) m [CR (CR2) PCR, H2N (CR2) P, RHN (CR2) P, HO (CR.!) P, HS (CR.!) P, halogen- (CR2) P, HOOC (CR2) P, ROOC (CR2) P, HCO (CR2) P, RCO (CR.!) P, or [HOOC (A) n) [ HOOC (B) m] (CR2) P, where p is O or integer from 1 to 20,

AYB are independently a substituted or unsubstituted Cj • alkyl, a substituted or unsubstituted C2 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted helerocyclic moiety, substituted or unsubstituted aromatic moiety.

According to the term C-alkyl, lO is understood to mean straight or branched chain alkyl groups having 1-10 carbon atoms, for example methyl, ethyl, isopropyl, butyl, and tert-butyl

The term C2.l0 alkenyl is understood to mean straight or branched chain alkenyl group having 2-10 carbon atoms, for example ethynyl, propenyl, isopropenyl, butenyl, and tert-butenyl.

According to the term cyclic moiety is understood as cyclohexane, and cyclopentane.

The term aromatic moiety is understood as phenyl.

The statement "A and B form a cyclic, heterocyclic or aromatic moiety ~ indicates a cyclohexane, piperidine, benzene, and pyridine.

In one embodiment, suitable dio, tri- or tetracarboxylic acids to be used in the present invention have the general formula

X [(A) nCOOH) [(B) mCOOH]

Where n and m are independently an integer from 1 to 20, X is HN, AYB are independently a substituted or unsubstituted C, .l0 alkyl, a substituted or unsubstituted C2_1 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted heterocyclic moiety or unsubstituted, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety In another embodiment, the acids gave, tri- or suitable tetracarboxylic acids to be used in the present method have the general formula

X [(A) nCOOH] [(B) mCOOH]

where n and m are O or an integer from 1 to 20, X is H2N (CR2) pCR, or RHN (CR2) pCR, where p is O or an integer from 1 to 20, where each R is H, a substituted Cj.jo alkyl or unsubstituted, a substituted or unsubstituted C2_l0 alkenyl, a moiety

10 substituted or unsubstituted cyclic, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety .

In yet another embodiment, suitable di, tri- or tetracarboxylic acids suitable for use in the present method have the general formula

0,.

'and

) X / (AI,

I

(Blm

'c' · 8

X [(A) nCOOH] [(B) mCOOH]

20 where n and m are O or an integer from 1 to 20, X is HO (CR2) pCR, HS (CR2) pCR, halogen- (CR2) pCR, HOOC (CR¡) pCR, ROOC (CR2) pCR, HCO (CR2) pCR, RCO {CR2) pCR, or [HOOC (A) n] [HOOC (B) m] CR (CR2) pCR, where p is O or integer from 1 to 20, each R independently is H or a Cl alkyl. 10 substituted or unsubstituted, a substituted or unsubstituted C2 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a moiety

25 substituted or unsubstituted aromatic, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety.

In another embodiment suitable di, tri- or tetracarboxylic acids suitable for use in the present method have the form general

30 0,.

'and

)

X / (AI,

)

IBlm

'C'

°

X [[A) nCOOHIIIB) mCOOH)

Where n and n are ° or an integer from 1 to 20, X is H2N (CR¡) P, RHN (CR2) P, HO (CR2) P, HS (CR; ¡) P, halogen- (CR2) P, HOOC (CR2) P, ROOC (CR2) P, HCO (CR2) P, RCO (CR2) P, or HOOC (A) n] [HOOC (B) m] (CR2) P, where p is O or integer of 1 at 20, each R independently is H or a substituted or unsubstituted Cl_l0 alkyl, a substituted or unsubstituted C2.l0 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety , or A and B together form a substituted or unsubstituted cyclic moiety, substituted heterocyclic moiety

40 or unsubstituted, substituted or unsubstituted aromatic moiety Di-, tri-Y tetracarboxylic acids favorable to provide the ring structure may be selected from imillO diacetic acid, 2-amino malonic acid, 3-amino glutaric acid, 3-methylamino acid glutaric acid, 3-chloro glutamic acid, 3-methoxycarbonyl glutaric acid, 3-acetyl glutaric acid, glutaric acid, tricarbalilic acid, 3,4-biscarboxymethyl adipic acid, 4- (2-carboxy-ethyl) -pyelic acid, (3 , 5-bis-carboxymethyl-phenyl) -acetic acid, 3,4-bis acid

5-carboxymethyl-adipic acid, benzene-1,2,4,5-tetra carboxylic acid, 4- (3-carboxy-allylamino) -but-2-enoic acid, 4,4imillO-dibenzoic acid, 1,4-dihydropyridine acid -3,5-dicarboxylic acid, 5-amino isophthalic acid, 2-chloro-malonic acid, 3-hydroxy glutaric acid, and benzene-1, 3,5-tricarboxylic acid

Fragment coupling (fragment coupling or fragment condensation) can be performed in

10 according to conventional procedures, for example as described in Peptide Synthesis protocols, Methods in Molecular Biology Vol. 35, Chapter 15, 303-3 16, Nyfeler R, Pennington MW and Dunne BM Eds_, Humana Press, 1994. Accordingly, The fragments can be synthesized in a solid phase, cleaved from the solid phase with complete preservation of protecting groups, purified and characterized as described above. Suitable fragments can also be obtained by other techniques described above.

According to the invention, two individual peptide sequences comprising two or more of the amino acid sequences defined above are connected to each other such that one of these two peptide sequences is covalent to one of two carboxylic groups of a linker molecule selected from The molecules defined above and another of the peptide sequences are covalently linked to another carboxylic group of said linker molecule. Peptide sequences covalently bind to the linker through their amino groups

or carboxy of the N-or C-terminal amino acid residue, respectively. Accordingly, a compound of the invention has the form

COOHfCONH2-peptide sequence-NH-CO-linker-CO-NH-peptide sequence-COOHfCONH2 25 or NH2-peptide sequence-CO-NH-linker-NH-CO-peptide sequence-NH2

In one embodiment, the compound according to the invention is exemplified by the following formula:

jVAENaQIJKf! f <A, CCli'ffl •.

neither! l (AENQQQI ($ I ("'~ NN;

Where TI is selected from the group you set in, where · is the junction point

• CH: .. "

"

: · Tl.

.b -, or ~~

In another preferred multimeric compound of the invention, two individual peptide sequences are connected between each other through two linkers or individual linker groups, such as

"-%you)

Linker 1. nlazador2 "Zf C

In lancer2 it can have the following structure where it indicates the point of attachment to the C-terminal end of the

Amino acid sequences Z1c or Linker2 is absent and the C-terminal group is an acidic acid or amide (unsubstituted amide shown in the structure).

, NHZ1bZ1 "" K "'MCONH,

!

; M! Z11lZ1a

Where 21 a is one or two amino acids independently selected from the group consisting of F, A, G, a, L, M, N, S, Y, T, 1, P; Vo is absent.

Z1b is an amino acid selected from the group consisting of F, A, G, O, L, M, N, S, Y, T, 1, P, V or is absent.

Z1d is a beta amino acid such as bela-alanine.

Each Z1c is independently selected from one of the peptide sequences shown in this document.

The e-terminal amide could be mono or disubstituted with a branched or unbranched C1-20 alkyl group. The alkyl group could be substituted with one or more functional groups such as a hydroxyl group, carboxylic acid group, an amide group or a halogen group

Linker1 may be, in one embodiment, a suitable diyral, tri- or telracarboxylic acid having the general formula '

where · indicates the point of attachment to the N-terminal of the amino acid sequences Z1c or Linker1 is absent and the N · terminal amino group is an NH2 group, where n and m are independently an integer from 1 to 20, X is HN, AYB are independently a substituted or unsubstituted alkyl 1.10, a substituted or unsubstituted alkenyl ~ .10, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted heterocyclic moiety

or unsubstituted, substituted or unsubstituted aromatic moiety

In another embodiment Linker1 is a suitable diiral, tri- or tetracarboxylic acid having the general formula:

where · indicates the point of attachment of the N-terminal of the amino acid sequence Z1c, where n and m are independently an integer from 1 to 20, X is Y1 b-Y1 a-N, A and B are independently a substituted alkyl and l_10

or unsubstituted, a substituted or unsubstituted e2.l0 alkenyl, a substituted or unsubstituted cyclic moiety, a moiety

Substituted or unsubstituted heterocyclic, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety

Where Y1 a is a spacer.

Where Y1 b is a lipophilic substituent comprising an acyl group of a straight or branched chain fatty acid

or a linear or branched dicarboxylic acid.

In one embodiment of the invention, the spacer is a dicarboxylic acid group having 1 to 7 methylene groups, such as two methylene groups, said spacer forming a bridge between the amino group to which Y1a is attached and a

Amino group of the lipophilic substituent In one embodiment of the invention, the spacer is selected from the list consisting of bela-alanine, gamma-amylobutyric acid (GASA), gamma-glutamic acid, Lys, Asp, Glu, a dipeptide containing Asp, a dipeptide containing Glu, or a dipeptide containing Lys. In one embodiment of the invention, the spacer is beta-alanine. In one embodiment of the invention, the spacer is gamma-aminobutyric acid (GASA). In an embodiment of the

5 invention, the spacer is quadrature-glutamic acid

In one embodiment of the invention, the amino group to which Y1a is attached forms an amide bond with a carboxylic group of a spacer, and an amino group of the spacer forms an amide bond with a carboxy group of the lip6file substituent.

In a further preferred embodiment, the lipophilic substituent is an acyl group of formula * C (C = O) - (CH) s..¡o-CH3 El * Specify the junction point. In a further preferred embodiment, the lipophilic substituent is an acyl group of formula * C (C = O) - (CH) s.26-CH3 El *

15 specifies the junction point. In a further preferred embodiment, the lipophilic substituent is an acyl group of formula * C (C = O) - (CH) s.20-CH3. The • specifies the junction point

In a further preferred embodiment, the lipophilic substituent is an acyl group of formula * C (C = O) - (CH) mCH3 * specifies the junction point month8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18.

In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid. In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid of formula * C (C = O) - (CH) 6- 4o-C (O) OH. The * specifies the junction point.

In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid of

formula * C (C = O) - (CH) S.26-C (O) OH. The • specifies the junction point. In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid of formula * C (C = O) - (CH) S.20-C (O) OH. The * specifies the junction point

In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid of formula * C (C = O) - (CH) S.S-C (O) OH. The * specifies the point of attachment In a further preferred embodiment, the lipophilic substituent comprises a linear or branched dicarboxylic acid of formula * C (C = O) - (CH)., - C (O) OH. The • specifies the junction point n eS8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. In another embodiment, Linker1 is a suitable achiral dio, tri- or tetracarboxylic acid having the formula general

where "indicates the junction point of the N-terminal part of the amino acid sequence Z1c, where n and m are Oo an integer from 1 to 20, X is H2N (CR2) pCR, or RHN (CR2) pCR, where p is Oo integer from 1 to 20, where each R is H, a substituted or unsubstituted C1_10 alkyl, a substituted or unsubstituted C2 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, an aromatic moiety substituted or unsubstituted, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted aromatic moiety

or without replacing.

In another embodiment, Linker1 is a suitable achiral, tri- or tetracarboxylic acid having the general structure where "indicates the point of attachment of the N-terminal part of the amino acid sequence Z1c, where n and m are O or an integer of 1 to 20, X is HO (CR2) pCR, HS (CR2) pCR, halogen- (CR.; ¡) PCR, HOOC (CR2) pCR, ROOC (CR2) pCR,

5 HCO (CR2) pCR, RCO (CR.; ¡) PCR, or [HOOC (A) n] {HOOC (B) m] CR (CR2) pCR, where p is O or integer from 1 to 20, each R it is independently H or a substituted or unsubstituted C_ "IO alkyl, a substituted or unsubstituted C2_10 alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together they form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety.

In another embodiment, Linker1 is a suitable di-, tri- or tetracarboxylic acid achiral having the general structure

15 X [(A) nCOOH] [(B) mCOOH] where "indicates the point of attachment of the N-terminal part of the amino acid sequence Z1c, where n and m are O or an integer from 1 to 20, X is H2N ( CRz) p, RHN (CR2) P, HO (CRz) p, HS (CR2) P, halogen- (CRz) p, HOOC (CR2) P, ROOC (CRup, HCO (CRúP, RCO (CR2) P, or [HOOC (A) n] [HOOC (B) m] (CR2) P, where p is O or integer from 1 to 20, each

R 20 is independently H or a substituted or unsubstituted Cl_lO alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety

In another embodiment, Linker1 is a suitable diiral, tri- or tetracarboxylic acid having the general structure

30 where ~ indicates the point of attachment of the N-terminal part of the amino acid sequence Z1c, where X is one of the following groups where · indicates the point of attachment According to the term alkyl el_lO is understood to mean straight or branched chain alkyl groups having 1 · 10 carbon atoms, for example methyl, ethyl, isopropyl, butyl, and tert-butyl.

The term "alkenyl" and "10" is understood to mean straight or branched chain alkenyl groups having 2-10 carbolyl atoms, for example ethynyl, propenyl, isopropenyl, butenyl, and tert-butenyl.

The term cyclic moiety is understood as cyclohexane, and cyclopentane.

The term "aromatic moiety" means phenyl.

The statement "A and 8 form a cyclic, heterocyclic or aromatic moiety" indicates cyclohexane, piperidine, benzene, and pyridine

By reaction with a carboxylic acid, a solid X-type (eO-sequence) 2-phase construction is obtained, where X is as defined above.

By the term "sequence" is meant, herein, a peptide comprising amino acids of natural origin and of non-natural origin, a PNA-sequence, or peptidomimetic. "Natural amino acids" means L and D forms of the 20 acids found in nature. Amino acids of unnatural origin are, for example, modified amino acids of natural origin. The term sequence further seeks to comprise a

or more of said sequences. Examples of suitable peptidomimetics are described in Marshall G.R., (1993) Tetrahedron, 49: 3547-3558. The term "chemical moieties" indicates an entity that enhances the solubility or biological activity of the LPA, and the entity to direct the LPA to its target or a marker. Preferred embodiments for the sequences have been described above.

Group X directly or indirectly allows continuous step synthesis or a coupling of fragments of the same sequence, or of one or more sequences and different moieties. The orientation of the peptridic fragments (N aeoea N) in LPA is defined as desired: In one embodiment, the present invention highlights LPA with N ae orientation, in another embodiment it refers to compounds with simultaneous N aeyea N presentation of the sequences , and in yet another embodiment, the sequences are oriented to N.

In the case where X comprises a temporarily protected amino function, the syntheses or coupling can be carried out directly after the protection. Proper activation of all carboxyl-containing groups that provide effective ring system formation (in step (c), see above) can be ensured using semi-equivalent carboxy acid. In the case of tri-or tetracarboxylic acids, the activated carboxy group can be further derivatized with a diamine such as ethylenediamine or an amine adequately functionalized for additional reactions such as mercapto, oxy, oxo or carboxyl groups. In the case of diamine, the synthesis of peptides or fragment coupling can be continued directly according to the desired sequence or chemical moiety. In a preferred embodiment, the Fmoc protection strategy is used, but any amino protection group can be used depending on the synthesis or coupling strategy Examples are the strategy with the protection group 80e.

As the continuous step synthesis or coupling of fragments is performed with one or in the case of a bifunctional chemical moiety such as lysine with two amino acid groups, it has surprisingly been discovered that a much better moiety can be obtained compared to conventional telomere lysine dendrimers obtained by the synthesis of MAP. In addition, optimal peptide synthesis procedures or coupling procedures for the individual chains bound to the solid phase can be used, and their homogeneity can be verified before forming the LPA. The solid phase cleavage and simultaneous deprotection of the side chain can be performed by conventional peptide synthesis procedures (described above). Therefore, a final product can be obtained that has an optimal and well defined composition. Puification can also be easily carried out by conventional chromatography methods such as HPLC or gel filtration, if desired or needed.

The favorable di ·, tri-Y tetracarboxylic acids to provide the ring structure can be selected from imillO diacetic acid, 2-amino malonic acid, 3-amino glutaric acid, 3-methylamino glutaric acid, 3-chloro glutamic acid, 3- methoxycarbonyl glutaric acid, 3-acetyl glutaric acid, glutaric acid, tricarbalilic acid, 3,4-biscarboxymethyl adipic acid, 4- (2-carboxy-ethyl) -pyelic acid, (3,5-bis-carboxymethyl-phenyl) -acetic acid , 3,4-biscarboxymethyl-adipic acid, benzene-1, 2,4,5-tetra carboxylic acid, 4- (3-carboxy-allylamino) -but-2-enoic acid, 4,4imillO-dibenzoic acid, 1 , 4-dihydropyridine-3,5-dicarboxylic acid, 5-amino isophthalic acid, 2-chloro-malophilic acid, 3-hydroxy glutaric acid, and benzene-1,3,5-tricarboxylic acid.

Fragment coupling (fragment coupling or fragment condensation) can be performed according to conventional procedures, for example as described in Peptide Synthesis protocols, Methods in Molecular Biology Vol. 35, Chapter 15, 303-316, Nyfeler R, Pennington MW and Ounne BM Eds., Humana Press, 1994. Accordingly, fragments can be synthesized in a solid phase, cleaved from the solid phase with complete preservation of the protecting groups, purified and characterized as described above. Suitable fragments can also be obtained. by other techniques described above

It is a preferred embodiment of the invention the use of the above method of LPA for the product: ion of a compound of the invention.

Medicine

An object of the invention is to provide a compound capable of modulating the activity of NCAM, said compound being referred to as a medicament for the treatment of diseases, where modulation of FGFR signaling can be considered as an essential condition for cure. .

Accordingly, the invention relates to the use of one or more of the peptides comprising a sequence corresponding to NCAM or a fragment thereof or a variant for the manufacture of a medicament.

In one embodiment, the medicament of the invention comprises at least one of the amino acid sequences set forth in SEQ ID NO: 1-4 or fragments or variants of said sequences. In another embodiment, the medicament of the invention comprises an antibody capable of binding to an epitope comprising NCAM or a fragment thereof or a fragment or variant of said antibody.

The medicament can prevent, in one aspect, the death of cells in vitro or in vivo, where the composition is administered to a subject, in vitro or in vivo in an effective amount of one or more of the compounds described above or a composition such as outlined below.

The medicament of the invention comprises an effective amount of one or more of the compounds defined above, or a composition comprising the compound defined above, in combination with pharmaceutically acceptable additives. Said medicament can be adequately formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonary administration.

Strategies in the development of drug formulations and compositions based on the peptides of the present invention generally correspond to formulation strategies for any other protein-based drug product. The potential problems and the guidelines required to overcome these problems are discussed in several textbooks, for example "Therapeutic Peptides and Protein Formulation. Processing and Oelivery Systems", Ed. AK. Banga, Technomic Publishing AG, Basel, 1995

Injectables are usually prepared as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid before injection: ion. The preparation can also be emulsified. The active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or enhancing the effectiveness or transport of the preparation.

Pharmaceutical compositions containing a compound according to the present invention can be prepared by conventional techniques, for example as described in Remington's Pharmaceutical Sciences, 1985 or in Remington: The Science and Practice of Pharmacy, 19th edition, 1995

In one embodiment of the invention, the pharmaceutical formulation is an aqueous formulation, that is, formulation comprising water. Said formulation is typically a solution or a suspension. In a further embodiment of the invention, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w of water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% pfp of water, and the expression "aqueous suspension" is defined as a suspension comprising at least 50% pfp of water.

An object of the present invention is to provide a pharmaceutical formulation comprising a compound according to the present invention that is present in a concentration of about 0.1 mgfml to about 50 mgfml, such as about 0.1 mgfml to about 25 mglml . The pH may be 2.0 to 10.0, such as 7.0 to 8.5. The formulation may additionally comprise a buffer system, preservative (s), isotonicity agent (s), chelating agent (s), stabilizers and surfactants. In another embodiment of the invention, the pH of the formulation is selected: ione from the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3, 9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6, 4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8, 9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. Preferably, the pH of the formulation is at least 1 unit of pH from the isoelectric point of the compound according to the present invention, even more preferably the pH of the formulation is at least 2 units of pH from the isoelectric point of the compound according with the present invention.

In another embodiment, the pharmaceutical phonulation is a freeze-dried formulation, to which the doctor or the patient adds solvents and / or diluents before use, to obtain, for example, the concentrations mentioned above.

In another embodiment, the pharmaceutical formulation is a dried formulation (for example, freeze dried or spray dried) ready for use without any prior dissolution.

In a further aspect, the invention relates to a pharmaceutical formulation comprising an aqueous solution of a compound according to the present invention, and a buffer, wherein said compound is present in a concentration of 0.1 mg / ml or above. , and where said formulation has a pH of about 2.0 to about 10.0

In a further aspect, the invention relates to a pharmaceutical formulation comprising an aqueous solution of a compound according to the present invention, and a buffer, wherein said compound is present in a concentration of 0.1 mg / ml or above. , and where said formulation has a pH of about 7.0 to about 8.5

The buffer may be selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium phosphate, and tris (hydroxymethyl} -aminomethane, hepesin, bicin , malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof Each of these specific buffers constitutes an alternative embodiment of the invention.

The formulation may further comprise a pharmaceutically acceptable preservative. In a further embodiment of the invention, the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, pcresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate , 2-Phenoxyethanol, butyl p-hydroxybenzoate, 2 phenylethanol, benzyl alcohol, ethanol, chlorobutanol, and thimerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzinephenophenophenophenophenophenochloride -1, 2-diol) or mixtures thereof. In a further embodiment of the invention, the preservative is present in a concentration of 0.1 mg / ml to 30 mg / ml. In a further embodiment of the invention, the preservative is present in a concentration of 0.1 mg / ml to 20 mg / ml. In a further embodiment of the invention, the preservative is present in a concentration of 0.1 mg / ml to 5 mg / ml. In a further embodiment of the invention, the preservative is present in a concentration of 5 mg / ml to 10 mg / ml. In a further embodiment of the invention, the preservative is present in a concentration of 10 mglml to 20 mg / ml. Each of these specific preservatives constitutes an actual alteration of the invention. The use of a preservative in pharmaceutical compositions is well known to experts. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19 '"edition, 1995

In a further embodiment of the invention, the formulation further comprises an isotonic agent. In a further embodiment of the invention, the isotonic agent is selected from the group consisting of a salt (for example, sodium chloride), a sugar or sugar alcohol, an amino acid (for example, Lg licin, L-histidine, arginine , lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (for example, glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol), polyethylene glycol (for example , PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water soluble glucans may be used, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pululane, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na. In one embodiment, the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one OH group and includes, for example, mannitol, sorbitol, inositol, galacititol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, provided that the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely affect the stabilizing effects achieved using the methods of the invention. In one embodiment, the concentration of sugar or sugar alcohol is between about 1 mg / ml and about 150 mgfml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 1 mg / ml to 50 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 1 mg / ml to 7 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 8 mg / ml to 24 mg / ml. In a further embodiment of the invention, the isotonic agent is present in a concentration of 25 mg / ml at 50 mglm. Each of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well known to experts. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19 "edition, 1995.

In a further embodiment of the invention, the formulation further comprises a chelating agent. In a further embodiment of the invention, the chelating agent is selected from salts of ethylenediamine tetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In a further embodiment of the invention, the chelating agent is present in a concentration of 0.1 ml ml to 5 ml ml. In a further embodiment of the invention, the chelating agent is present in a concentration of 0.1 mglml to 2 mglml. In a further embodiment of the invention, the chelating agent is present in a concentration of 2 mglml to 5 mglml. Each of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well known to the experts. Remington: The Science and Practice of Pharmacy, 19 "edition, 1995 is convenient for convenience.

In a further embodiment of the invention, the formulation further comprises a stabilizer. The use of a stabilizer in pharmaceutical compositions is well known to those skilled in the art. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

More particularly, the compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly shows aggregate formation during storage in liquid pharmaceutical formulations. By "formation of aggregates ~ a physical interaction between polypeptide molecules that causes the formation of oligomers, which may remain soluble, or large visible aggregates precipitating in the solution is intended. For ~ during storage ~ a pharmaceutical composition or formulation is intended liquid that once prepared, is not immediately administered to a subject.Instead, after preparation, it is packaged for storage, in a liquid form, in a frozen state, or in a dried form for subsequent reconstitution in a liquid form or another suitable form for administration to a subject. By "dried form" it is intended that the liquid pharmaceutical composition or formulation be dried by freeze-drying (ie, lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38: 48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (S "ed; Longman Scientific and Technic al, Essez, R.U.), p. 491-676; Broadhead et al (1992) Drug Devel. Ind. Pharm. 18: 1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11: 12-20), or air dried (Carpenter and Crowe (1988) Cryobiology 25: 459-470; and Roser (1991) Biopharm. 4: 47-53). The formation of aggregates by a polypeptide during storage of a liquid pharmaceutical composition may adversely affect the biological activity of that polypeptide, causing the loss of therapeutic efficacy of the pharmaceutical composition. In addition, the formation of aggregates can cause other problems such as blockage of tubes, membranes, or pumps when the pharmaceutical composition containing polypeptide is administered using an infusion system.

The term "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term "physical stability" of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and insoluble aggregates of the protein as a result of the exposure of the protein to thermomechanical stresses and interaction with surfaces. of contact and surfaces that are destabilizing, such as hydrophobic surfaces and contact surfaces. The physical stability of aqueous protein formulations is assessed by visual inspection and turbidity measurements after exposing the filled formulation in suitable containers (e.g., cartridges or vials) to mechanical-physical stress (e.g., agitation) at different temperatures for various periods. of time. The visual inspection of the formulations is done in a focused and sharp light with a dark background. The turbidity of the formulation is characterized by classification in visual value of the degree of turbidity, for example, on a scale of O to 3 (a formulation that does not show turbidity corresponds to a visual value O, and a formulation that shows visual turbidity to the Daylight corresponds to visual value 3). A formulation is classified as physically unstable with respect to protein aggregation when it shows visual turbidity in daylight. Alternatively, the turbidity of the formulation can be evaluated by simple turbidity measurements well known to the experts. The physical stability of aqueous protein formulations can also be evaluated using a spectroscopic agent or probe of the conformational state of the protein. The probe is preferably a small molecule that preferentially binds to a non-native protein conformer. An example of a small molecular spectroscopic probe of the protein structure is Tioflavin T. Tioflavin T is a ftuorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps also protein configurations as well, Tioftavin T gives rise to a new excitation maximum at approximately 450 nm and enhanced emission at approximately 482 nm when bound to a protein form of fibril. Unbound thioflavin T is essentially non fluorescent at wavelengths

Other small molecules can be used as probes for changes in protein structure from native states to non-native states. For example, "hydrophobic patch" probes that preferably bind to exposed hydrophobic patches of a protein. Hydrophobic patches are usually buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature. Examples of these small molecular spectroscopic probes are hydrophobic aromatic colors, such as anthracene, acridine, phenanthroline or the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.

The term "chemical stability" of the protein formulation as used herein refers to chemical covalent changes in the protein structure that lead to the formation of chemical degradation products with potential lower biological potency and / or potential increased immunogenic properties in comparison with the native protein structure. Various chemical degradation products may be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. The elimination of chemical degradation most likely cannot be completely avoided and increasing amounts of chemical degradation products are often observed during storage and use of the protein formulation as is well known to those skilled in the art. Most proteins are prone to deamidation, a process in which the amide group of the glutaminyl or asparaginyl moiety is hydrolyzed to form a free carboxylic acid. Other degradation pathways involve the formation of high molecular weight transformation products where two or more protein molecules covalently bind to each other through transamidation and / or disulfide interactions that lead to the formation of dimeric, oligomeric and polymeric covalently bound degradation products. (Stability of Protein Pharmaceuticals, Ahern. TJ YManning MC, Plenum Press, New York 1992). Oxidation (for example methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein formulation can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions (the formation of degradation products can often be accelerated, for example, by increasing the temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on the size and loading of the molecule using various chromatography techniques (eg, SEC-HPLC and / or RP-HPLC).

Therefore, as summarized above, a "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation must be stable during use and storage (in accordance with the recommended conditions of use and storage) until the expiration date is reached.

In one embodiment of the invention, the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 6 weeks of use and for more than 3 years of storage.

In another embodiment of the invention, the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of use and for more than 3 years of storage.

In a further embodiment of the invention, the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of use and for more than two years of storage.

In a further embodiment of the invention, the pharmaceutical formulation comprising the compound is stable for more than 2 weeks of use and for more than two years of storage. The pharmaceutical compositions of the invention may additionally comprise an amount of an amino acid base sufficient to decrease the formation of aggregates by the polypeptide during storage of the composition By "amino acid base" an amino acid or a combination of amino acids is intended, wherein any This given amino acid is present in its free base form or in its salt form. When a combination of amino acids is used, all amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their forms. Salinas. In one embodiment, the amino acids used to prepare the compositions of the invention are those that carry a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid. In one embodiment, the amino acid used to prepare the compositions of the invention is glycine. Any stereoisomer (i.e., L or D) of a particular amino acid (eg, methionine, histidine, imidazole, arginine, lysine, isoleucine, acid) may be present. aspartic, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, in the pharmaceutical compositions of the invention provided that the particular amino acid is present in its free base form or its salt form. In one embodiment the stereoisomer L is used. The compositions of the invention can also be formulated with analogs of these amino acids. By "amino acid analog" is intended a derivative of the naturally occurring amino acid that achieves the desired effect of decreasing the formation of aggregates by the polypeptide during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogs include, for example, aminoguanidine, omitin and N-monoethyl L-arginine, suitable methionine analogs include ethionine and butionin and suitable cysteine analogs include S-methyl-L cysteine. Like the other amino acids, amino acid analogs are incorporated into the compositions in their free base form or in their salt form. In a further embodiment of the invention, amino acids or amino acid analogs are used in a concentration that is sufficient to prevent or delay the aggregation of the protein.

In an additional embodiment of the invention, methionine (or other amino acids or sulfuric amino acid analogs) may be added to inhibit the oxidation of methionine residues in methionine sulfoxide when the polypeptide that acts as a therapeutic agent is a polypeptide comprising at least one moiety. of methionine susceptible to such oxidation. By "inhibiting" the minimum accumulation of oxidized methionine species over time is intended. Inhibition of methionine oxidation causes greater retention of the polypeptide in its appropriate molecular form. Any methiooine stereoisomer (L, O or a mixture thereof) can be used. The amount to be added must be an amount sufficient to inhibit the oxidation of methionine residues so that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine so that the ratio of methionine added to the methionine moieties ranges from about 1: 1. at about 1000: 1, such as from 10: 1 to about 100: 1

In a further embodiment of the invention, the formulation further comprises a stabilizer selected from the group of high molecular weight polymers or low molecular weight compounds. In a further embodiment of the invention, the stabilizer is selected from polyethylene glycol (for example, PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxyhydroxycellulose or derivatives thereof (for example, HPC, HPC- SL, HPC-L and HPMC), cyclodexlrins, sulfur-containing substances such as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (for example, sodium chloride). Each of these specific stabilizers constitutes an alternative embodiment of the invention.

The pharmaceutical compositions may also comprise additional stabilizing agents, which further enhance the stability of a therapeutically active polypeptide therein. Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a non-ionic surfactant, which protects the polypeptide against aggregation associated with freeze-thaw or breakage. mechanics.

In a further embodiment of the invention, the formulation further comprises a surfactant. In a further embodiment of the invention, the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolized glycerides, acetylated moooglycerides, sortJitan fatty acid esters, polymers of polyoxypropylene-polyoxyethylene block (for example, poloxamers such as Pluronic.RTM. F68, poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters, star-shaped PEO, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, for example Tween-20, Tween-40, Tween-BO and Brij-35), polyoxyethylene hydroxystearate, moooglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol , lecithins and phospholipids (for example, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inosyl ol, diphosphatidyl glycerol and sphingomyelin), phospholipid derivatives (for example, dipalmitoyl phosphatidic acid) and lysophospholipids (for example, palmitoyl lysophosphatidyl-L-serine and 1-acyl-sn-glycerol-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxy (alkyl ester), alkoxy (alkyl ether) derivatives of lysophosphatidyl and phosphatidylcholines, for example lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, ie hills, ethanolamines, acid phosphatidic, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidyltreonine, and glycerophospholipids (for example, cephalins), glyceroglycolipids (for example, galactopylid (galactopyliphoids, galactopylides (galactopylides) gangliosides), dodecylphosphocholine, chicken egg lysolecitin, fusidic acid derivatives - (for example, taurus dihydrof sodium usidate, etc.), long chain fatty acids and salts thereof C6-C12 (for example, oleic acid and caprylic acid), acylcamitins and derivatives, N.sup .. alpha-acylated derivatives of lysine, arginine or histidine , or acylated derivatives in the lysine or arginine side chain, N.sup .. alpha-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, derivative N.sup ..alpha. - acylation of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (sodium docusate, CAS registration no (577-11-7)), calcium docusate, GAS registration no (128-49-4 ]), potassium docusate, CAS registration number (7491-09-0]), SOS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, colic acid or derivatives thereof, bile acids and salts thereof and conjugates thereof glycine or taurine, ursodeoxycholic acid, sodium colalate, sodium deoxycholate, tau sodium rocolate, sodium glycocholate, N-hexadecyl-N, N-dimethyl3-ammonium-1-propanesulfonate, anionic monovalent surfactants (alkyl-aryl sulphonates), zwitterionic surfactants (eg, N-alkyl-N, N-dimeti lammoo-) 1-propanosu Ifonates, 3-colamido-1-propild imetyl ammonium-1propanesulfonate), cationic surfactants (quatematic ammonium bases) (for example, cetyltrimethylammonium bromide, cetylpyridinium chloride), nonionic surfactants (eg, Dodecyl. Dodecyl. -D-glucopyranoside), poloxamines (for example, Tetronic), which are tetrafunctional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant can be selected from the group of imidazoline derivatives, or mixtures thereof. Each of these specific surfactants constitutes an alternative embodiment of the invention,

The use of a surfactant in pharmaceutical compositions is well known to experts. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

It is possible that other ingredients may be present in the pharmaceutical peptide formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, volume agents, tonicity modifiers, chelating agents, metal ios, oleaginous vehicles, proteins (for example, human serum albumin, gelatin or proteins) and a zwitterion (for example, a amino acid such as betaine, taurine, arginine, glycine, lysine and histidine) _ Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

The compositions of the invention can be further compounded in, or linked to, for example, through covalent, hydrophobic and electrostatic interactions, a drug vehicle, drug delivery system and advanced drug delivery system to further enhance the stability of the compound, increase bioavailability, increase solubility, decrease adverse effects, achieve well-known chronotherapy for those skilled in the art, and increase patient cooformity or any combination thereof. Examples of vehicles, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol, polymers of acrylate and methacrylate, polylacrylic and polyglycolic acid and block copolymers thereof, polyethylene glycols, vehicle proteins, for example albumin, gels, for example, thermogelling systems, for example block copolymer systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and dispersions thereof, phase L2 and dispersions thereof, well known to those skilled in the art of phase behavior in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifiers , self-emulsifiers, cyclodexlrins and derivatives thereof, and dendrimeros

The compositions of the present invention are specifically useful in the formulation of controlled, sustained, prolonged, delayed, and slow-release drug delivery systems_ More specifically, the compositions are useful in the formulation of parenteral systems of controlled tiberation and sustained release ( leading both systems to a reduction in many times the number of administrations), well known to those skilled in the art. Even more preferably, either the controlled release and sustained release systems administered subcutaneously. They are limiting the scope of the invention, examples of useful controlled release systems and compositions are hydrogels, oil gels, liquid crystals, polymeric micelles, microspheres, nanoparticles.

Methods for producing controlled release systems useful for compositions of the present invention include, but are not limited to, crystallization, co-condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Cootrolled Release (Wise, D_ L, ed _Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99 · Protein Formulation and Delivery (MacNally, E_J _, ed_Marcel Dekker, New YOfk, 2000)

Thus, injectable compositions of the GLP-1 derivative of the invention can be prepared using conventional techniques of the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired final product.

According to one procedure, the GLP-1 derivative is dissolved in an amount of water that is somewhat smaller than the final volume of the composition to be prepared. An isotonic agent, a co-preservative and a buffer are added as necessary and the pH value of the solution is adjusted - if necessary - using an acid, for example hydrochloric acid, or a base, for example aqueous sodium hydroxide according to necessary. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

In addition to the components mentioned above, solutions containing a GLP-1 derivative according to the present invention may also contain a surfactant to improve the solubility and {or stability of the GLP-1 derivative.

A composition for nasal administration of certain peptides can be prepared, for example, as described in European Patent No. 272097 (de Novo Nordisk AlS) or in document W093118785

In accordance with a preferred embodiment of the present invention, the GLP-1 derivative is provided in the form of a composition suitable for administration by injection. Such composition may be an injectable solution ready for use or may be an amount of a solid composition, for example, a lyophilized product, which has to be dissolved in a solvent before it can be injected_ The injectable solution preferably contains not less than about 2 mg / ml, preferably not less than about 5 mgfml, more preferably not less than about 10 mg {ml of the GLP-1 derivative and, preferably, no more than about 100 mg / ml of the GLP-1 derivative.

Formulations of the compounds of the invention can be prepared by techniques known to those skilled in the art. Formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like as described above.

Pharmaceutical compositions containing the compound according to the present invention can be administered to a patient in need of such treatment at various sites, for example, in topical sites, for example, skin and mucous sites, at sites that elude absorption, by for example, administration in an artery, in a vein, in the heart, and at sites that involve absorption, for example, administered in the skin, under the skin, in a muscle or in the abdomen

The administration of pharmaceutical compositions according to the invention can be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for example, through the conjunctiva, urethral, and parenteral to patients who need such treatment

The compositions of the present invention can be administered in various dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, ointments, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example , hard gelatin capsules and soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution , solutions for on-site transformation, for example on-site gelation, on-site fixation, on-site precipitation, on-site crystallization, infusion solution, and implants.

The compositions of the present invention are useful in the formulation of solids, semi-solids, powder and solutions for pulmonary administration of the compound using, for example, a metered dose inhaler, dry powder inhaler and a nebulizer, I feel all well known devices for experts in the field.

Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by a syringe, optionally a pen syringe. Alternatively, parenteral administration can be performed by an infusion pump. An additional option is a composition that can be a solution or suspension for the administration of the compound according to the present invention in the form of a nasal or pulmonary spray. As a further additional option, pharmaceutical compositions containing the compound of the invention can also be adapted for transdermal administration, for example, by needleless injection or from a patch, optionally an iontophoretic patch, or transmucosal administration, for example, oral.

other formulations are equally suitable for nasal and pulmonary administration, for example, inhalers and aerosols

The active compound can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid. Salts formed with the free carboxyl group can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine

The preparations are administered in a manner compatible with the dosage formulation, and in an amount such that it will be therapeutically effective. The amount to be administered depends on the subject to be treated including, for example, the weight and age of the subject, the disease to be treated and the stage of the disease. Suitable dosage ranges are per kilogram of body weight normally on the order of several hundred g of active ingredient per administration with a preferred range of about 0.1 I1g to 5000 g per kilogram of body weight using monomeric forms of the compounds , suitable dosages are often in the range of 0.1 ~ 5000 I1g per kilogram of body weight, such as in the range of about 0.1 I1g to 3000 I1g per kilogram of body weight, and especially in the range of approximately 0.1 I1g to 1000 I1g per kilogram of body weight. Using multimeric forms of the compounds, suitable dosages are often in the range of O, 1 ~ g to 1000 I1g per kilogram of body weight, such as in the range of approximately O, 1 ~ g to 750 I1g per kilogram of body weight, and especially in the range of about 0.1 I1g to 500 I1g per kilogram of body weight such as in the range of about 0.1 ~ g to 250 ~ g per kilogram of body weight. In particular when administered nasally, smaller dosages are used than when administered by other routes. Administration may be performed once or may be followed by subsequent administrations. The dosage will depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosage of multimeric forms would range from 1 mg to 70 mg per 70 kg of body weight. For most indications, a localized or substantially localized application is preferred.

Some of the compounds of the present invention are sufficiently active, but for some of the olros, the effect will be enhanced if the preparation additionally comprises pharmaceutically acceptable additives and / or vehicles. Such additives and vehicles will be known in the art. In some cases, it will be advantageous to include a compound, which promotes the supply of the active substance to its target as described above.

In many cases, it will be necessary to administer the formulation multiple times. Administration may be a continuous infusion, such as infusion or intraventricular administration in more doses such as more times a day, daily, more times a week, weekly, elc. It is preferred that the administration of the drug be initiated before or after the individual has undergone the factor or factors that can lead to cell death. Preferably, the medicament is administered in 8 hours from the appearance of the factor, such as in 5 hours from the appearance of the factor. Many of the compounds show a long-term effect whereby the administration of the compounds can be done with long intervals, such as 1 week or 2 weeks. In relation to the use in nerve guides, administration can be continuous or in small parts based on the controlled release of the active compound or compounds. In addition, precursors can be used to control the release rate and / or release site. Other types of implants as well as oral administration may also be based on controlled release and / or the use of precursors.

As discussed above, the present invention relates to compounds for use in a treatment of individuals to induce differentiation, modulate proliferation, stimulate regeneration, neuronal plasticity and cell survival in vitro or in vivo, involving treatment. administer an effective amount of one or more compounds defined above

Another strategy for administration is to implant or inject cells capable of expressing and secreting the compound in question. That way the compound can be produced at the location where it will work.

Treatment

The compounds according to the invention are particularly useful for treating neurodegenerative diseases, as well as conditions with altered cognition, and the treatment according to the invention is, in one embodiment, useful for inducing differentiation, modulating proliferation, stimulating regeneration, Neural plasticity and cell survival, for example cells that are implanted or transplanted. The compounds are useful for the diseases and conditions mentioned below, in particular, useful for the treatment of A1zheimer's disease, Parkinson's disease, Huntington's disease, cerebral ischemia, as well as stress

In a further embodiment, the treatment may be for stimulation of the survival of cells that are at risk of dying due to a variety of factors, such as trauma and injury, acute diseases, chronic diseases and disorders, in particular degenerative diseases that normally lead to cell death, other external factors, such as medical and / or surgical treatments and diagnostic methods that can cause free radical formation or otherwise have cytotoxic effects, such as X-rays and chemotherapy. In relation to chemotherapy, the peptides according to the invention are useful in the treatment of cancer.

Therefore, the treatment includes the treatment and prophylaxis of cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, for example resulting from spinal cord injury, altered fiber myelination nerve, postischemic damage, for example resulting from a stroke, multi-infarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington's disease.

In addition, in relation to diseases or conditions of the muscles, including conditions with altered function of neuro-muscular connections, such as genetic or traumatic trophic disorders of the muscles; or for the treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas, such as diabetes mellitus type I and 11 of the kidney, such as nephrosis, the compounds according to the invention can be used to induce the differentiation, modulate proliferation, stimulate regeneration, neuronal plasticity and survival, that is, stimulate survival.

In addition, treatment may be to prevent cell death of heart muscle cells, such as after acute myocardial infarction, to induce angiogenesis. In addition, in one embodiment, the treatment is for the stimulation of survival of heart muscle cells, such as survival after acute myocardial infarction. In another aspect, the treatment is for revascularization, such as after injuries

Also within the scope of the invention is a use of the peptides to promote wound healing. The present peptides are capable of stimulating angiogenesis and thus can promote the wound healing process.

The invention further describes a use of peptides in the treatment of cancer.

Regulation of tyrosine kinase receptor activity is important for tumor angiogenesis, proliferation and spread.

In a further embodiment, a use of the peptides is for the stimulation of the ability to learn yfo from short and long term memory, since the activity of FGFR is important for the differentiation of neural cells

In yet another embodiment, a peptide for use according to the invention is for the treatment of bodily harm due to alcohol consumption. It concerns particularly malformations in the development of fetuses, long-term neurobehavioral disorders, alcoholic liver disease

The therapeutic treatment of prion diseases that includes the use of a peptide is yet another embodiment of the invention.

In particular, the use according to the invention of a peptide may be for the treatment of clinical conditions, such as malignancies such as malignant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain behavior, breast cancer, thyroid, pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ, muscles (sarcoma), cancers with dysfunction, and overexpression or sub-expression of specific receptors and expression of mutated receptors or associated with soluble receptors, such as recipients of Ero and FGF receptors, endocrine gland diseases, such as diabetes mellitus I and 11, pituitary gland tumor, psychosis, such as senile and presenile organic psychotic conditions, alcoholic psychosis, pOf drug psychosis, transient organic psychotic conditions, Alzheimer's disease , cerebral lipidosis, epilepsy, general paresis {syphilis], hepatolenticular degeneration, korea of Huntington, Jakob-Creulzfeldt disease, multiple sclerosis, Pick's disease of the brain, polyarteritis nadosa, syphilis, schizophrenic disorders, affective psychosis, neurotic disorders, personality disorders, including characteristic neurosis, non-psychotic personality disorder associated with organic brain syndromes , paranoid personality disorder, fanatic personality, paranoid personality (disorder), paranoid traits, sexual deviations and disorders or dysfunctions (including reduced sexual desire for any reason), mental retardation, nervous system disease and sensory organs, such as disability of vision, hearing, smell, touch, taste, cognitive abnormalities after illness, injury (for example, after trauma, surgical procedure, and violence), inflammatory disease of the central nervous system, such as meningitis, encephalitis, brain degenerations such as disease of Alzheimer's, Pick's disease, senile degeneration of the brain, unspecified senility, communicating hydrocephalus, obstructive hydrocephalus, including Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie Sanger Brown, cerebellar myoclonic dysinergia, primary cerebellar degeneration , such as spinal muscular atrophy, familial, juvenile, spinal muscular atrophy in the adult, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, progressive bulbar paralysis, pseudobulbar paralysis, primary lateral sclerosis, other anterior horn cell diseases , anterior horn cell disease, unspecified, other spinal cord diseases, syringomyelia and syringobulbia, vascular myelopathies, acute spinal cord (embolic) infarction (non-embolic), spinal cord arterial thrombosis, spinal cord edema, hematomyelia, myelopa Subacute necfotic aunt, subacute combined degeneration of the spinal cord in diseases classified elsewhere, myelopathy, drug-induced myelitis, radiation-induced, autonomic nervous system disorders, autonomic, sympathetic, parasympathetic, or vegetative peripheral system disorders, familial dysautonomy [ Riley-Day syndrome], idiopathic peripheral autonomic neuropathy, syncope and carotid sinus syndrome, cervical sympathetic dystrophy or paralysis, peripheral autonomic neuropathy in disorders classified elsewhere, amyloidosis, peripheral nervous system diseases, brachial plexus lesions, cervical rib, costclavicular syndrome, anterior scalene syndrome, thoracic operculum syndrome, brachial neuritis or unspecified radicu lithis, including in neonates, inflammatory and toxic neuropathy, including acute infectious polyneuritis, Guillain-Barre syndrome, post-i polyneuritis Infectious, polyneuropathy in collagen vascular disease, balloon disorders including disorders that affect multiple structures of the eye, such as purulent endophthalmitis, mastoid ear and apophysis diseases, chronic rheumatic heart disease, ischemic heart disease, arrhythmia, pulmonary system diseases, system respiratory, sensory, for example, oxygenation, asthma, abnormality of organs and soft tissues in neonates, including in the nervous system, complications of the administration of anesthesia or other sedation during labor and delivery, skin diseases including infection, problem of insufficient circulation, burn injury and other mechanical and physical injuries, injuries, including after surgery, crush injury, burns, nerve and spinal cord injuries, including nerve division, continuity injury (with or without open wound), traumatic neuroma (with or are open wound), to transient traumatic lysis (with or without open wound), accidental perforation or laceration during medical procedure, injury to the optic nerve and pathways, injury to the optic nerve, second cranial nerve, injury to the optic chiasma, injury to the optic pathways, injury to the visual cortex, unspecified blindness, injury to another nerve or cranial nerves, injury to other and unspecified nerves, drug poisoning, medicinal and biological substances, genetic or traumatic atrophic muscle disorders; or for the treatment of diseases or conditions of various organs, such as degenerative diseases of the gonads, of the pancreas, such as diabetes mellitus type I and 11, of the kidney, such as nephrosis, Scrapie, CreulzfeldtJakob disease, Gerstmann-Straussler disease- Sheinker (GSS); pain syndrome, encephalitis, drug / alcohol use, anxiety, postoperative nerve damage, peri-operative ischemia, inflammatory disorders with tissue damage, affecting the infectious agent or protecting the tissue, HIV, hepatitis, and the following symptoms, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS, anti-inflammatory effects, asthma and other allergic reactions, acute myocardial infarction, and other related disorders or AMI sequences, metabolic disorders, such as lipid disorders of obesity (for example, hypercholesterolemia, artherosderosis, transport disorders and amino acid metabolism, purine and pyrimidine metabolism disorders and gout, bone disorders, such as fracture, osteoporosis, osteoarthritis (DA), atrophic dermatitis, psoriasis, disorders caused by infection, protection or maturation of stem cells in vivo or in vitro.

In a further embodiment, the medication is administered to increase well-being, such as stimulation of sexual motivation, desire or pleasure. Well-being can be physical as well as psychological.

Antibody

An objective of the present description provides the use of an antibody, antigen-binding fragment or recombinant protein thereof capable of selectively binding an epitope comprising a contiguous amino acid sequence derived from NCAM or a fragment, homologue or variant thereof. . The description refers to any antibody capable of selectively binding to an epitope comprising a contiguous amino acid sequence derived from NCAM, selected from any of the sequences set forth in SEQ ID NOS 14, or a fragment or variant of said sequence.

The term "epitope" means the specific group of atoms (in an antigen molecule) that the antibodies (of that antigen) recognize. The term "epitope" is the equivalent of the term "antigenic determinant." The epitope may comprise 3 or more amino acid residues, such as 4, 5, 6, 7, 8 amino acid residues, located in close proximity, such as within an amino acid sequence of contiguous ones, or located in distant parts of the amino acid sequence of an antigen, but which due to protein folding have approximated each other.

The antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic essential component, the folding or immunoglobulin domain, is used in various forms in many molecules of the immune system and other biological recognition systems. A typical immunoglobulin has four polypeptide chains, which contain an antigen-binding region known as a variable region and a non-variable region known as a constant region.

Native antibodies and immunoglobulins are usually heterotetramerican glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, although the amount of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intracatenary disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end: The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form a counting surface between the light and heavy chain variable domains (Novotny J, and Haber E Proc Natl Acad Sci USA. 82 (14): 4592-6, 1985).

Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins · IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), for example IgG-1, IgG-2, IgG-3 and IgG4 ; IgA-1 and IgA-2. The constant domains of the heavy chains that correspond to the different classes of immunoglobulins are called alpha (a), delta (o), epsilon (E), gamma (y) and mu (1-1), respectively. The light chains of the antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A-), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "variable" in the context of variable antibody domain refers to the fact that certain parts of the variable domains differ widely in sequence between antibodies. The variable domains are for binding and determine the specificity of each particular antibody by its particular antigen. However, the variability is not evenly distributed across the variable domains of the antibodies. It focuses on three segments called complementarity determining regions (CDRs) also known as hypervariable regions in the variable domains of both the light chain and the heavy chain.

The most highly conserved parts of the variable domains are called f1anquentes regions (FR)

The variable domains of native heavy and light chains each comprise four FR regions, which largely adopt a sheet configuration a, co-operated by three CDRs, which form connection loops, and in some cases form part of the sheet structure . The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibodies. Constant domains are not directly involved in the binding of an antibody to an antigen, but show various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present disclosure may therefore be in any of a variety of forms, including a complete immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody that includes the complementarity determining regions (CDR) of the variable domain, all of which are under the broad term "antibody, as used herein. The present description contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limits to antibodies that recognize and immuno-react with a specific antigen In the context of both therapeutic and screening methods described below, preferred embodiments are the use of an antibody or fragment thereof that is immunospecific for an antigen or epitope of the invention.

The term "antibody fragment" refers to a part of a full-length antibody, generally the antigen-binding or variable region. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments. Digestion with antibody papain produces two identical antigen-binding fragments, called a Fab fragment, each with a single antigen-binding site, and a residual "Fe" fragment, so called for its ability to crystallize rapidly. Pepsin treatment produces an F (ab ') 2 fragment that has two antigen-binding fragments that have cross-binding capacity to the antigen, and a different residual fragment (called pFc'). Additional fragments may include diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, "functional fragment" with respect to antibodies refers to Fv, F (ab) and F (ab), fragments.

The term "antibody fragment" is used interchangeably herein with the expression "antigen binding fragment"

The antibody fragments can be as small as about 4 amino acids, S amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino acids, about 15 amino acids, about 17 amino acids, about 18 amino acids, about 20 amino acids, about 25 amino acids, approximately 30 amino acids or more. In general, an antibody fragment can have any upper size limit as long as it has similar or immunological properties with respect to the antibody that specifically binds an epitope comprising a peptide sequence selected from any of the sequences identified herein as SEQ ID. NO 1-13, or a fragment of said sequences. Therefore, in the context of the present description, the expression "antibody fragment" is identical to the expression "antigen binding fragment".

Antibody fragments retain some ability to selectively bind with their antigen or receptor. Some types of antibody fragments are defined as follows:

Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of the complete antibody with the papain enzyme to produce an intact light chain and a part of a heavy chain.

Fab 'is the fragment of an antibody molecule that can be obtained by treating the complete antibody with pepsin, followed by reduction, to produce an intact light chain and a part of the heavy chain. Two Fab 'fragments are obtained per antibody molecule.

Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxyl end of the heavy chain CH1 domain that includes one or more cysteines of the hinge region of the antibody

(Fab ') 2 is the fragment of an antibody that can be obtained by treating the complete antibody with the enzyme pepsin without further reduction.

F (ab ') 2 is a dimer of two Fab' fragments held together by two disulfide bonds

Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain and a light chain variable domain in an adjusted non-covalent association (VH-Vd dimer. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-V dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind to the antigen, although at a lower affinity than the entire binding site

Single chain antibody ("SCA") is defined as a genetically engineered molecule that contains the variable region of the light chain, the variable region of the heavy chain, joined by a suitable polypeptide linker as a genetically fused single chain molecule . Such single chain antibodies

They are also referred to as ~ single chain Fv ~ or "sFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies 113: 269-315 Rosenburg and Moore eds. Springer-Verlag, NY, 1994

The term "diabody" refers to small antibody fragments with two antigen binding sites, said fragments comprising a heavy chain variable domain (VH) connected to a variable light chain (VL) domain in the same polypeptide chain ( VH-VL.) Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. more fully described in, for example, EP 404,097; WO 93111161, and Hollinger et al., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).

The description also contemplates multivalent antibodies that have at least two binding domains. Binding domains can have specificity for the same ligand or for different ligands. In one embodiment, the multispecific molecule is a bispecific antibody (BsAb), which at least two different binding domains, at least one of which is of antibody origin. Multivalent antibodies can be produced by several methods. Various methods for preparing bi-or multivalent antibodies are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; Y

5,482,858

The description contemplates both polyclonal and moncclonal antibodies, antigen-binding fragments and recombinant proteins thereof that are capable of binding to an epitope according to the invention.

The preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al. 1992, Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1

The preparation of monoclonal antibodies is also conventional. See, for example, Kohler and Milstein, Nature, 256: 495-7 (1975); Coligan, et al., Sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: A Laboratory Manual, page 726, Cold Spring Harbor Pub. (1988). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well established techniques. Such isolation techniques include Protein-A Sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography. See, for example, Coligan, et al., Sections 2.7.1-2.7.12 and sections 2.9.12.9.3; Bames, et al Purification of Immunoglobulin G (lgG). In: Methods in Molecular Biology, 1992, 10: 79-104, Humana Press, NY

The methods of in vitro and in vivo manipulation of monoclonal antibodies are well known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256, 495-7, or they can be prepared by recombinant methods, for example, such as described in US 4,816,567

Monoclonal antibodies for use with the present disclosure can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624-628, as well as in Marks et al., 1991, J Mol Bio1222: 581-597. The air method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing specific human and recognizable sequences. See, for example, Holmes, et al., 1997, J ImmunoI158: 2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol 81: 105-115

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts Monoclonal antiaBodies are highly specific, being directed against a single antigenic site, and in contrast to conventional polyclonal antibody preparations that

Typically they include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclooal antibodies are advantageous because they are synthesized by hybridoma culture, not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and should not be understood to require antibody production by any particular method.

The monoclonal antibodies herein specifically include chimeric (immunoglobulin) antibodies "in which a part of the heavy and light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody , while the rest of the chain or chains is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subdase of antibody, as well as fragments of said antibodies, provided they show the desired biological activity (document US 4,816,567); Morrison et al., 1984, Proc Natl Acad Sci 81. 6851-6855.

Methods for preparing antibody fragments are also known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1988). The antibody fragments of the present description can be prepared by proteolytic hydrolysis of the antibody or by expression in

E. coli of the DNA encoding the fragment. Antibody fragments can be obtained by conventional methods of digestion with pepsin or papain of whole antibodies. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment designated F (ab ') 2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for sulfhydryl groups resulting from cleavage of disulfide bonds, to produce monovalent 3.5 S Fab 'fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab 'fragments and one Fe fragment directly. These methods are described, for example, in US 4,036,945 and US 4,331,647, and references contained therein

Other methods can also be used to cleave antibodies, such as heavy chain separation to form monovalent fragments of light-heavy chain, additional cleavage of fragments, or other enzymatic, chemical, or genetic techniques, provided that the fragments bind to the antigen that the intact antibody recognizes. For example, Fv fragments comprise an association of VH and VL chains. This association may be non-covalent or the variable chains may be linked by an intermolecular disulfide bond or cross linked by chemical agents such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single chain antigen-binding proteins (sFv) are prepared by constructing a structural gene that comprises DNA sequences encoding the VH and VL domains connected by an oligonucleatide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide connecting the two V domains. Methods for producing sFv are described, for example, pOf Whitlow, et al., 1991, In: Methods: A Companion to Methods in Enzymology, 2 : 97; Bird et al., 1988, Science 242: 423-426; US 4,946,778; and Pack, et al., 1993, BioTechnology 11: 1271-77.

Another form of an antibody fragment is a peptide that encodes a single complementarity determining region (COR). COR peptides Minimum recognition units ·) are often involved in recognition and antigen binding. CDR peptides can be obtained by cloning or constructing genes encoding the COR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region of the antibody-producing cell RNA. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991)

The description contemplates human and humanized forms of non-human antibodies (eg, murine). Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antibody antigen binding sub-sequences) that contain a minimal sequence derived from non-immunoglobulin. human, such as the epitope recognition sequence. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the remains of a recipient complementarity determining region (COR) are replaced by residues of a COR of a non-human species (donor antibody) such as mouse, rat or rabbit that has the specificity, affinity and capacity desired. The humanized antibody or antibodies containing one or more minimum antibody sequences or antibodies of the description, such as one or more sequences that recognize one or more epitopes described herein, is one of the preferred embodiments of the invention.

In some cases, f1anqueante Fv residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies may comprise residues that are neither found in the recipient antibody nor in the sequences imported from CDR or nanquentes. These modifications are made to further refine and optimize the functioning of the antibody. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence of human immunoglobulin. The humanized antibody will also optimally comprise at least a part of a constant region of immunoglobulin (Fe), typically that of a human immunoglobulin. For additional details, see: Jones et al., 1986, Nature 321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992, Curr Op Struct BioI2: 593596; Holmes et al., 1997, J Immunol 158: 2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol. 81: 105-115.

Antibody generation can be achieved by any conventional method in the art for producing polydonal and monodonal antibodies using natural or recombinant fragments of a sequence selected from any of the sequences identified as SEQ ID NO: 1-4, as an antigen. Such antibodies can also be generated using variants or fragments of SEQ ID NO: 1-4

The antibodies can also be produced in vivo by the individual to be treated, for example, by administering an immunogenic fragment according to the invention to said individual. Accordingly, the present description further relates to a vaccine comprising an immunogenic fragment described above.

The description also relates to a method for producing an antibody of the invention, said method comprising a step of providing an immunogenic fragment described above.

The description refers both to an antibody that is capable of modulating, such as enhancing or attenuating, the function

10 of NCAM, in particular a function related to the growth and survival of neural cells, such as an antibody that can specifically recognize and bind to NCAM without modulating its biological activity.

The description refers to the use of the above antibodies for therapeutic applications that involve the modulation of NCAM activity.

In one aspect, the description refers to the use of a pharmaceutical composition comprising an antibody described above.

SEQUENCE LIST

<110> ENKAM

<120> New peptides derived from NCAM (FGLS)

<130> P1767PCOO

<160> 73

<170> Patentln version 3.5

<210> 1 <211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Derived from NCAM

<400> 1

val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing

1 5 10

<210> 2 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 2

Glu val val Ala 'Glu Asn Gln Gln Gl y l yS' ~ ér tys Ala 1 5 · 10

<210> 3 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 3

ASA Val val Wing Glu Asn Gln Gln Gly LyS .se r l ys Wing 1 5 10

<210> 4 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 4

Asn Se r val Glu Wing Asn Gln Gl n Gly Lys Ser Lys Ala 1 5 lQ

<210> 5 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 5

Wing val Wing Glu Asn Gln Gln Gly LyS Ser Lys Wing 1 S 10

<210> 6 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 6

Gly val Wing Glu Asn Gln Gln GlY Lys be Lys Wing 1 S 10

<210> 7 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 7

Leu val Ala Glu Asn Gln Gln Gly Lys Ser LyS Ala 1 S 10

<210> 8 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 8

Met val Ala Glu Asn Gln.Gln Gly l ys Ser lys Ala 1 S 10

<210> 9 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 9

Val val Wing Glu Asn Gln Gln Gly LyS Ser Lys Wing 1 S 10

<210> 10 <211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 10

pro val Al a Gl u Asn Gln Gln Gly Lys Ser Lys Ala 1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 11

rle val Ala Glu Asn Gln Gln Gly l ys Ser lys Ala

1 5 W

<210> 12 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 12

Gln Allah Val Wing Glu Asn Gln Gln ', Gly-lyS Ser lYS Ala

one . 5 10

<210> 13 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 13

Gln Gly Val Wing Glu Asn Gln Gln Gly lys Ser lys Wing 1 5 10

<210> 14

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 14

Gln leu val Allah Glu Asn Gln Gln Gly Lys Ser Lys Ala 1 5 10

<210> 15

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 15

Gln Met Val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing 1 S 10

<210> 16

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 16

Gln val val Wing Glu Asn Gln Gln Gly Lys be Lys Wing 1 S 10

<210> 17

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 17

Glñ Pro Val Wing Glu Asn Gln Gln G] and'LYS being Lys Wing 1 "S 10

<210> 18 <211> 13

<212> PRT <213 »Artificial sequence

<220>

<223> counterpart

<400> 18

Gln ¡le val Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala

1 5 10

<210> 19

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 19

Asn Ala val Ala Glu Asn Gln Gln Gly LyS be LyS Ala 1 S 10

<210> 20

<211> 13

<212> PRT

<213> Artificial sequence

<220> <223> counterpart

<400> 20

ASO 'Gly Val Wing Glu "Asn Gln Gln Gly Lys Ser lys Wing 1 5 10 ·

<210> 21 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 21

Asn leu val Ala Glu Asn Gln Gln Gly lys Ser lys Ala 1 5 10

<210> 22

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 22

Asn Met val Wing Glu Asn Gln Gln Gly lys''ser ly.s Wing 1 S ro

<210> 23

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 23

Asn Val val Glu Wing Asn Gl "Gln Gly lyS Ser lys Ala 1 5 la

<210> 24 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 24

Asn Pro val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing l · S 10

<210> 25 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 25

Asn il @ Val Ala Glu'Asn Gln Gln Gly Lys Ser · ~ Lys Ala

1 .. S 10

<210> 26

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 26

Ser Ala val Ala Glu Asn Gln Gln Gly lys Ser Lys Ala 1 5 10

<210> 27 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 27

Be Gly Val Wing Glu Asn Gln Gln Gly Lys Be Lys Wing 1 5 10

<210> 28 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 28

Be Leu Val Ala Glu Asn Gln Gln Gly Lys ser_Lys Ala 1 S 10

<210> 29

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 29

Ser Met val Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala 1 5 10

<210> 30

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 30

Ser Val Val. Ala Glu Asn Gln Gln Gl and LyS Ser l.ys Al a

1 · 5 10

<210> 31

<211> 13 5 <212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 31

Ser Pro val Ala Glu Asn Gln Gln Gly Lys Ser lys Ala

5·

1 10

15 <210> 32 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 32

be rle val Wing G] U Asn Gln Gln Gly Lys Ser Lys Al a 25 1 5 10

<210> 33 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

35 <400> 33

lh.r Wing val Wing Glu Asn Gln Gln Gly lys Ser Lys Wing 1 5 10

<2 10> 34 <211> 13

<212> PRT

<213> Artificial sequence

<220> 45 <223> counterpart

<400> 34

Thr Gly val Ala Glu Asn Gln Gln Gly Lys be LyS Ala 1 5 10

<210> 35 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 35

Thr leu val Ala Glu Asn Gln Gln Gly Lys Ser lys Ala 1 5 10

<210> 36 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 36

ihr Met val Ala Glu Asn Gln Gln Gly Lys Ser lys Ala

1 · 5 10

<210> 37

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 37

Thr 'val val Ala Glu As n Gln Gln Gly LyS Ser Lys, Ala 1 S 10

<210> 38 <211> 13

<212> PRT <: 213> Artificial sequence

<220> <: 223> counterpart

<400> 38

Thr Pro val Ala Glu Asn Gln Gln Gly lys Ser l ys Ala 1 5 10

<210> 39 <: 211> 13

<212> PRT <: 213> Artificial sequence

<: 220>

<223> counterpart

<400> 39

Thr ¡le val Ala Glu Asn Gln Gln Gl and Lys Ser Lys Al at 1 5 10

<210> 40 <: 211> 13 <: 212> PRT

<213> Artificial sequence <: 220>

<223> counterpart

<400> 40

Gly Wing val Wing Glu Asn Gln Gln Gly LyS Ser Lys Wing 1 5 10

<210> 41

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 41

Gly Gly Val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing 1 S 10

<210> 42

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 42

Gly Leu Val Wing Glu Asn Gln Gln Gly Lys be Lys Wing 1 S 10

<210> 43

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 43

Gly Met val Al to Glu Asn Gln Gln Gly Lys be LyS Ala 1 S 10

<210> 44

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 44

Gly val val Ala Glu Asn Gln Gl n Gly ly_s Ser l ys Ala 1 S 10

<210> 45

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 45

Gly Pro val 1

Glu wing 5 Asn Gln Gln Gl and lys Ser Lys "Ala 10

<210> 46

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 46

Gly Ile Val Ala Glll Asn Gln Gln, Gly l ys Ser lys Ala 1 5 10

<210> 47

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 47

ASP Wing val Wing GJll Asn Gln Gln Gly lyS Ser Lys Wing 1 5 10

<210> 48

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 48

ASp Gly: val A'la Glu Asn Gln Gln -Gly Lys -Ser. ~ Ys A.la l '5 10

<210> 49

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 49

ASp l "eu Val Ala Gl ll Asn Gln Gln Gly Lys se'r Lys Ala 1 5 10

<210> 50

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 50

Asp Met val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing 1 5 10

<210> 51

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 51

ASp val val Ala Glu Asn Gln Gln Gly lys Ser Lys A.la

1 S "10

<210> 52

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 52

ASP pro val Wing Glu Asn Gln Gln Gly LyS be LyS Wing 1 5 10

<210> 53 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 53

ASp rle val Ala Glu As "Gln" Gln Gly lys s: er lys Ala

1 5 10

<210> 54

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 54

Glu Ala val Ala Glu Asn Gln Gln Gly l ys Ser l ys Ala 1 5 10

<210> 55

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 55

Glu Gly val Ala Glu Asn Gln · Gln Gly lys Ser lys Ala i 5 10

<210> 56

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 56

í lU Leu Val Al a ~ lu Asn Gln, Gln Gly ibs Ser Lys Ala

<210> 57

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 57

Glu Met Val Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala 1 5 10.

<210> 58 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 58

Glu Pro val Ala Glu Asn Gln Gln Gly lys Ser Lys Ala 1 5 10

<210> 59 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 59

Glu Ile val Ala Glu Asn Gln Gln Gly LyS be LyS Ala

1 5 10

<210> 60 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 60

'Wing ASp val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing 1 5 10

<210> 61 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 61

C:;: ly "ASp val" Ala Glu Asn Gln Gln Gly Lys Ser Lys Ala

1 5 10

<210> 62 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 62

Leu ASp Val Ala Glu Asn Gln "Gln Gly ~ ys Ser Lys Ala 1 5 10

<210> 63 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 63

Mét ASp Val Ala Glu Asn "Gln Gln Gly Lys Ser Lys Ala 1 5 10

<210> 64

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 64

val ASp val Wing Glu Asn Gln Gln Gly LyS Ser LyS Wing 1 S 10

<210> 65

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 65

Pro ASp Val Wing Glu Asn Gln Gln Gly Lys Ser Lys Wing 1 S 10

<210> 66

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 66

11 e ASp val Ala A'sn Gl n Gln Gly Lys Ala

G1 "LY 'Se r

1 5 10

<210> 67 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 67

Wing Glu Val Wing Glu Asn Gln Gln Gly LyS Ser Lys Wing 1 5 10

<210> 68

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 68 Gly Glú val Ala Glu As "G1n Gln Gly Lys Ser Lys Ala 1 5 lU

<210> 69 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 69

Leu Glu Val Wing G1 u Asn · Gln Gln Gly Lys Ser LyS Wing 1 5 10

<210> 70 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 70

Me 'Glu val Ala Glu. Asn Gln Gln Gly Lys Ser LyS Ala 1 5 10

<210> 71 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 71

ra1 Glu val Al y ~ Asn Gln Gln Gly iós Ser lys Ala

<210> 72 <211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 72

Pro Glu Val Ala Glu Asn Gln Gln Gly lys ser lys Ala 1 5 10

<210> 73

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> counterpart

<400> 73

Ile Glu val Ala Glu Asn Gln Gln Gly lyS be LY.S Ala 1 5 10

Claims (12)

A peptide of 13 amino acids or less comprising the sequence QQGKSKA, or a pharmaceutically acceptable salt thereof. 2. A peptide according to claim 1, wherein said peptide is selected from the group consisting of VAENQQGKSKA (SEQ ID No. 1) 10 EWAENQQGKSKA (SEQ ID No. 2) NWAENQQGKSKA (SEQ ID No. 3) and NSVAENQQGKSKA (SEQ ID No. 4), or a fragment or variant thereof, wherein said variant comprises at least one amino acid substitution. 3. A compound consisting of a dimer of two peptides according to claim 1 or 2 connected to each other through a linker or linker group, wherein said compound is selected from the group consisting of: O) VAENOOGKSKA-CON ~ "N ~ VAEN ~ KSKA-CONH ~ O ~ EVVAENCK) GKSKA ~ NH2 HN YEVVAENOOGKSKA'CONH! O ~~ AENCKX3KSKA ~ HI "N andNWAENOOGKSKA'CON ~ ° rNSVAENOOGKSKA'9QNH¡ HH and NSVAENQQGKSKA'CONHz 4 A compound according to claim 3, wherein said compound is' . 0) VAENQOGKSKA · CQNH, "N ~ VAENCKlGKSKA-CONHt 5. A peptide according to claim 1 or 2 or a compound according to claim 3 or 4, wherein said peptide is capable of interacting with an FGFR, such as binding to FGFR.

6.

A peptide or a compound according to claim 5, wherein said peptide is capable of modulating FGFR signaling, stimulating FGFR signaling and inhibiting FGFR signaling.

7.

A peptide or a compound according to any one of the preceding claims, wherein said peptide is capable of stimulating neurite spread, cell survival, synaptic plasticity, learning and memory.

8.

A pharmaceutical composition comprising at least one peptide or compound defined in any of the preceding claims.

9.

A peptide or a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use as a medicament.

10.

A peptide or a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in the treatment of diseases or conditions in which modulation of FGFR signaling is essential.

eleven.

A compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in the treatment of diseases or conditions of the central or peripheral nervous system.

12.

A compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in the treatment of a disease or condition in which the stimulation of neural cell differentiation, the survival of neural cells , neurogenesis, stem cell proliferation, stem cell differentiation, and / or learning and memory is beneficial for the recovery of said disease or condition.

13.

A compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in the treatment of stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, postoperative nerve damage, traumatic nerve damage , altered myelination of nerve fibers, post-ischemic damage, multi-infarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, mood disorders, or manic depressive disorders.